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WO2025194315A1 - Nucleic acid detection method and use thereof - Google Patents

Nucleic acid detection method and use thereof

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Publication number
WO2025194315A1
WO2025194315A1 PCT/CN2024/082289 CN2024082289W WO2025194315A1 WO 2025194315 A1 WO2025194315 A1 WO 2025194315A1 CN 2024082289 W CN2024082289 W CN 2024082289W WO 2025194315 A1 WO2025194315 A1 WO 2025194315A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
nucleic acid
region
fragment
detection probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/082289
Other languages
French (fr)
Chinese (zh)
Inventor
王毅雄
杨梦�
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MGI Tech Co Ltd
Original Assignee
MGI Tech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MGI Tech Co Ltd filed Critical MGI Tech Co Ltd
Priority to PCT/CN2024/082289 priority Critical patent/WO2025194315A1/en
Publication of WO2025194315A1 publication Critical patent/WO2025194315A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to the field of nucleic acid hybridization technology, and in particular to a nucleic acid detection method and a combined detection method thereof with protein detection.
  • Fluorescence digital pathology microscopy is a key technology in the life sciences. By combining state-of-the-art omics methods with fluorescence microscopy and digitized parameters, biological samples (such as tissue sections) can be visualized digitally in millions of pixels, displaying tens of thousands of features.
  • a rapid, multiplexed digital pathology immunofluorescence instrument has been developed. Based on a desktop series of sequencers, it now supports tissue protein fluorescence staining and imaging, in addition to basic sequencing capabilities. Currently, this instrument focuses on multiple rounds of protein staining and photography on tissue sections. However, in potential future digital pathology diagnostic applications, multiple rounds of protein staining can only be considered a single diagnostic basis, leaving significant room for improvement. Therefore, adding the capture and identification of RNA hybridization signals at the target site to multiple rounds of protein staining can further enhance the accuracy of digital pathology diagnoses.
  • the existing multiple immunofluorescence technology needs to block the sample at room temperature, and each round of antibody elution needs to be re-blocked, which increases the staining time.
  • Traditional immunofluorescence staining takes a long time, about 210 minutes, and the traditional staining method lacks a multiple immunofluorescence detection method for a single slice.
  • the iterative indirect immunofluorescence imaging method is an improved version of the traditional method. After imaging, an antibody eluent (containing TCEP, urea, guanidine hydrochloride, Tris-HCl) is added for 30 minutes of antibody elution and then the staining step is repeated.
  • an object of the present invention is to provide a nucleic acid detection primer combination or suit, a nucleic acid hybridization method, a method for detecting a target nucleic acid, and a method for nucleic acid, protein joint detection (joint detection) etc.
  • the incubation time of fluorescence reading/detection nucleic acid (such as RNA) hybridization signal can be greatly shortened, and it is ensured that after the nucleic acid molecule signal hybridization, multiple rounds of protein staining are continuously realized on the sample (such as tissue), i.e., a nucleic acid (such as RNA)-protein joint detection method, which can provide dual evidence of nucleic acid level and protein level to the target target staining by the detection result.
  • a nucleic acid (such as RNA)-protein joint detection method which can provide dual evidence of nucleic acid level and protein level to the target target staining by the detection result.
  • first primers include: a fourth region that pairs and binds to a fragment of a target nucleic acid, and at least one first region that does not pair and bind to the target nucleic acid;
  • the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid;
  • a third primer comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid.
  • the present invention utilizes the described tertiary primer combination to ensure that the three primers have complementary binding domains. Furthermore, under the action of a ligase, the primer sequences at each level can be looped in a timely manner, further enabling tight binding and exponential amplification of the three primers.
  • This unique primer sequence design based on the tertiary primer hybridization logic, allows for the targeted capture of hybridization signals, significantly shortening the incubation time for fluorescent reading/detection of target nucleic acid hybridization signals. For example, whereas traditional fluorescence in situ hybridization techniques require overnight incubation at 37°C in a humid environment, a similarly efficient reaction can now be completed in only about 90 minutes.
  • the primer combination of the present invention can be applied to in situ hybridization. After the probe fluorescent substance is eluted, multiple rounds of protein staining can be continued on the tissue to provide double evidence for the staining of the target point.
  • the first primer includes: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that bind to the target nucleic acid sequence without pairing.
  • the second region does not pair-bind with the first region.
  • the fourth region includes a first fragment and a second fragment, the first fragment and the second fragment are respectively located at the two ends of the first primer, and the first region is located between the first fragment and the second fragment; each first region includes at least a third fragment and a fourth fragment; the fifth region includes a fifth fragment and a sixth fragment, the fifth fragment and the sixth fragment are respectively located at the two ends of the second primer, and the second region is located between the fifth fragment and the sixth fragment; each second region includes at least a seventh fragment and an eighth fragment; the sixth region includes a ninth fragment and a tenth fragment, the ninth fragment and the tenth fragment are respectively located at the two ends of the third primer, and the third region is located between the ninth fragment and the tenth fragment; each third region includes at least the third fragment and the fourth fragment.
  • the primer combination includes a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid.
  • the first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the fragment of the target nucleic acid to which they are paired and bound.
  • the lengths of the first fragment and the second fragment are each independently 15-20 nt.
  • the lengths of the third fragment and the fourth fragment are each independently 8-20 nt, preferably 10-16 nt.
  • the lengths of the fifth fragment and the sixth fragment are each independently 8-20 nt, preferably 10-16 nt.
  • the lengths of the seventh fragment and the eighth fragment are each independently 8-20 nt, preferably 10-16 nt.
  • the lengths of the ninth fragment and the tenth fragment are each independently 8-20 nt, preferably 10-16 nt.
  • the first primer includes more than one first region, and the first regions are connected by a first spacer sequence.
  • each of the second regions is connected by a second spacer sequence.
  • each of the third regions is connected by a third spacer sequence.
  • the length of the first spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.
  • the length of the second spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.
  • the length of the third spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.
  • the second aspect of the present invention provides a kit, which comprises: the primer combination described in the first aspect, and a first detection probe and/or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of the region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.
  • the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.
  • the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.
  • the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.
  • the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.
  • the label comprises a radioactive label or a non-radioactive label.
  • the non-radioactive label comprises a fluorescent group.
  • a third aspect of the present invention provides a nucleic acid hybridization method, comprising the following steps:
  • step S2 contacting the product obtained in step S1 with the second primer in the primer combination described in the first aspect, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain a target nucleic acid-first primer-second primer hybrid 2;
  • step S3 the product obtained in step S2 is contacted with the third primer in the primer combination described in the first aspect, and the sixth region of the third primer is paired and bound with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3.
  • the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.
  • the method further includes: before performing step S2, using a ligase to connect the first primer present in the system after the reaction of step S1 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess first primers that are not bound to the target nucleic acid.
  • the method further includes: before performing step S3, using a ligase to connect the second primer present in the system after the reaction of step S2 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess second primers that are not bound to the first primer.
  • the method further includes: after performing step S3, using a ligase to connect the third primer present in the system after the reaction of step S3 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess third primers that are not bound to the second primer.
  • the method further comprises: performing a ligation reaction using the ligase at a reaction temperature of 30-40° C. and a reaction time of 5-30 min.
  • the ligase is T4 ligase.
  • the cleaning solution comprises ethylene carbonate, and the concentration of ethylene carbonate in the cleaning solution is 5-40% v/v.
  • the cleaning solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.
  • the reaction temperature for pairing and binding in step S1 is 30-40° C.
  • the reaction time is 30-120 min.
  • the reaction temperature for the pairing binding in step S2 is 30-40° C.
  • the reaction time is 20-40 min.
  • the reaction temperature for the pairing binding in step S3 is 30-40° C.
  • the reaction time is 20-40 min.
  • the method further comprises:
  • step S3 continue to repeat steps S2-S3 for at least N rounds, where N ⁇ 1 and N is an integer; or,
  • step S3 continue to repeat steps S2-S3 for at least N' rounds, N' ⁇ 0, N' is an integer, and only repeat step S2 in the last round.
  • the fourth aspect of the present invention provides a method for detecting a target nucleic acid, the method comprising: using the nucleic acid hybridization method described in the third aspect to obtain a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer, and detecting the hybrid using a first detection probe and/or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of a region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of a region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.
  • the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.
  • the first detection probe comprises at least one of the seventh fragments in the second primer.
  • the sequence of the first portion and the eighth segment is paired and bound to at least a portion of the first portion.
  • the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.
  • the second detection probe comprises a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.
  • the target nucleic acid is single-stranded DNA or RNA.
  • the method further comprises: after step S2, detecting a hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe.
  • the method further includes: after step S3, continuing to repeat steps S2-S3 for at least N rounds, detecting the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer with the second detection probe, and optionally, using the first detection probe for auxiliary detection, where N is a natural number.
  • steps S2-S3 are repeated for at least N' rounds, and in the last round, only step S2 is repeated.
  • the first detection probe is used to detect the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer, and optionally, the second detection probe is used for auxiliary detection, wherein N' is a positive integer.
  • the lengths of the first and second detection probes are independently 13-23 nt.
  • a fifth aspect of the present invention provides a method for combined detection of nucleic acids and proteins, comprising the following steps:
  • the protein fluorescent staining comprises performing multiple rounds of protein staining using protein markers (eg, antibodies).
  • protein markers eg, antibodies
  • the sixth aspect of the present invention provides uses of the primer combination described in the first aspect and the kit described in the second aspect in the following:
  • the biological sample to be tested in the combined detection of nucleic acids and proteins, is preferably fixed on a solid support.
  • the biological sample to be tested is a tissue/cell section sample.
  • This invention improves upon traditional in situ hybridization staining methods, significantly increasing staining efficiency and shortening staining time. It is also applicable to conventional manual staining systems, improving tissue section staining efficiency, reducing operator workload, and shortening working hours.
  • This method has a wide range of potential applications, including tissue mapping, RNA in situ hybridization, and aptamer staining. It can be used to construct maps of plant and animal cell distribution, tumors, and diseases, as well as for digital pathology and biomarker screening and discovery.
  • the inventors used the primer combination described above to perform fluorescence in situ hybridization, aiming to increase the capture and identification of nucleic acid molecule hybridization signals of the target target under the premise of multiple rounds of protein staining in the fast and multiple digital pathology immunofluorescence instrument, so as to realize the joint detection of nucleic acids and proteins on the machine.
  • the experimental design includes tissue section preparation (dewaxing and hydration antigen repair), nucleic acid hybridization molecule sequence design and preparation, nucleic acid hybridization process reagent preparation, protein multiple rounds of staining reagent preparation, and integrated automatic staining and shooting of the entire process.
  • the multiple rounds of hybridization of nucleic acid molecules in tissue sections, amplification, fluorescence signal reading, and fluorescence signal elution are added to achieve the effect of joint detection of nucleic acids and proteins.
  • primer #1 the first primer in the primer combination of the present invention
  • primer #2 the second primer
  • primer #3 the third primer
  • the primer combination provided in the present invention is applied to the joint detection of nucleic acids and proteins in biological samples (such as tissue sections).
  • the nucleic acid and protein automated dyeing and shooting machine ensures the repeatability of position information for background correction and rapid antibody incubation.
  • the nucleic acid and protein joint detection in the present invention can be completely formed into an automated dyeing and shooting platform through a sequencer.
  • FIG1 is a schematic diagram showing the amplification process when performing RNA fluorescence detection using a first primer, a second primer, and a third primer;
  • Figure 2 shows the comparative results of fluorescence in situ hybridization of the target CD8 mRNA under different reaction conditions in Example 1, wherein A. fluorescence in situ hybridization results under different read probe concentrations; B. statistical results of fluorescence in situ hybridization under different read probe concentrations;
  • Figure 3 shows the comparative results of fluorescence in situ hybridization of the target CD8 mRNA in Example 2 in a 10% ethylene carbonate reaction system (left) and a 10% polyvinylamine reaction system (right);
  • FIG4 shows the results of tissue protein fluorescence staining of the target proteins PD-L1 and CD8 protein in Example 3.
  • first and second are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as “first” or “second” may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, “plurality” means two or more.
  • the terms “optionally,” “optional,” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • primer refers to an artificially synthesized oligonucleotide sequence that is complementary to at least a portion of a template strand of a target region.
  • the primer may be a single-stranded DNA primer or an RNA primer.
  • the present invention provides a primer combination comprising:
  • first primers include: a fourth region that pairs and binds to a fragment of a target nucleic acid, and at least one first region that does not pair and bind to the target nucleic acid;
  • the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid;
  • a third primer comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid.
  • the first primer in the primer combination of the present invention can be designed as a plurality of different first primer sequences, and these different sequences can be complementary paired with different segments of the target nucleic acid, respectively. After the first primer binds to the target nucleic acid, it further hybridizes with the second primer and the third primer to form multiple target nucleic acid-primer hybrids.
  • the primer combination of the present invention can be applied to in situ hybridization. After the probe fluorescent substance is eluted, multiple rounds of protein staining can be continued on the tissue to provide double evidence for the staining of the target point.
  • the first primer includes: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that do not pair and bind to a sequence of the target nucleic acid.
  • the second region does not pair with the first region; and the third region does not pair with the second region.
  • the primer combination of the present invention it is ensured that the second region does not pair and bind with the first region.
  • Such primer design can ensure that the fifth region of the second primer and the second region do not competitively bind with the first region of the first primer.
  • the third region does not pair and bind with the second region.
  • Such primer design can ensure that the third region and the sixth region of the third primer do not competitively bind with the second region of the second primer.
  • the fourth region includes a first fragment and a second fragment, the first fragment and the second fragment are respectively located at the two ends of the first primer, and the first region is located between the first fragment and the second fragment; each first region includes at least a third fragment and a fourth fragment; the fifth region includes a fifth fragment and a sixth fragment, the fifth fragment and the sixth fragment are respectively located at the two ends of the second primer, and the second region is located between the fifth fragment and the sixth fragment; each second region includes at least a seventh fragment and an eighth fragment; the sixth region includes a ninth fragment and a tenth fragment, the ninth fragment and the tenth fragment are respectively located at the two ends of the third primer, and the third region is located between the ninth fragment and the tenth fragment; each third region includes at least the third fragment and the fourth fragment.
  • the primer combination includes a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid.
  • the primer combination provided by the present invention when designed to include: a first segment and a second segment complementary to fragments of different target nucleic acids, and at least two first regions that do not bind to the sequence of the target nucleic acids, can be used to simultaneously detect multiple target nucleic acids.
  • the first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the target nucleic acid fragments to which they bind.
  • the first base at the 5' end of the first primer and the last base at the 3' end correspond to adjacent bases on the target nucleic acid fragment with which they are paired and bound. This means that when the first primer is paired and bound to the target nucleic acid fragment, the 5' end and 3' end of the first primer are respectively paired and bound to the target nucleic acid fragment, and the middle segment (first region) is not bound.
  • the first primer forms a ring structure, and the first base at the 5' end of the first primer is adjacent to the last base at the 3' end, which facilitates subsequent ligation into a ring by a ligase.
  • the primer combination provided by the present invention includes:
  • first primers include: a fourth region that binds to a target nucleic acid fragment, and two first regions that do not bind to the target nucleic acid;
  • the second primer comprising: a fifth region that pairs and binds to the first region, and two second regions that do not pair and bind to the target nucleic acid;
  • a third primer comprising: a sixth region that pairs and binds to the second region, and two third regions that do not pair and bind to the target nucleic acid.
  • the length of the first fragment and the second fragment are each independently 15-20 nt.
  • the length of the first fragment and the second fragment are each independently 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt.
  • the length of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment is each independently 8-20 nt, preferably 10-16 nt.
  • the length of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment is each independently 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt or 16 nt.
  • the first primer includes more than one first region, and the first regions are connected by a first spacer sequence.
  • the provision of the first spacer sequence makes it easier for the second primer to bind to the first primer.
  • each of the second regions is connected by a second spacer sequence.
  • the provision of the second spacer sequence makes it easier for the third primer to bind to the second primer.
  • each of the third regions is connected by a third spacer sequence.
  • the provision of the third spacer sequence makes it easier for the second primer in the next round to bind to the third primer.
  • the length of the first spacer sequence, the second spacer sequence and the third spacer sequence is independently at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.
  • the length of the first spacer sequence, the second spacer sequence and the third spacer sequence is each independently 2 nt, 3 nt, 4 nt or 5 nt.
  • the present invention also provides a kit comprising: the primer combination described above, and a first detection probe and/or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of the region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.
  • the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.
  • the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.
  • the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.
  • the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.
  • the label comprises a radioactive label or a non-radioactive label.
  • the non-radioactive label comprises a fluorescent group.
  • the present invention also provides a nucleic acid hybridization method, comprising:
  • step S2 contacting the product obtained in step S1 with the second primer in the aforementioned primer combination, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain target nucleic acid-first primer-second primer hybrid 2;
  • step S3 the product obtained in step S2 is contacted with the third primer in the primer combination described above, and the sixth region of the third primer is paired and bound with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3.
  • the nucleic acid hybridization method further comprises: the contact in steps S1-S3 is carried out in a hybridization reaction solution containing ethylene carbonate, wherein the concentration of ethylene carbonate in the hybridization reaction solution is 5- 40% v/v, preferably, the concentration of ethylene carbonate in the hybridization reaction solution is 10% v/v.
  • the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.
  • the hybridization reaction solution is 10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, and the solvent is 1X SSC.
  • the nucleic acid hybridization method further includes: before performing step S2, using a ligase to connect the first primer present in the system after the reaction of step S1 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess first primers that are not bound to the target nucleic acid.
  • the nucleic acid hybridization method further includes: before performing step S3, using a ligase to connect the second primer present in the system after the reaction of step S2 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess second primers that are not bound to the first primer.
  • the nucleic acid hybridization method further includes: after performing step S3, using a ligase to connect the third primer present in the system after the reaction of step S3 is completed to form a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess third primers that are not bound to the second primer.
  • the temperature and time of the ligation reaction is preferably carried out at a temperature suitable for the ligase, which may fluctuate above or below the optimal temperature for ligation.
  • the ligation time only needs to ensure that all single-stranded primers are ligated into a ring.
  • the nucleic acid hybridization method further comprises: using the ligase to perform a ligation reaction at a reaction temperature of 30-40° C. and a reaction time of 5-30 minutes.
  • the reaction temperature is 37° C. and the reaction time is 20 minutes.
  • the reaction temperature for the pairing binding in step S2 is 30-40° C.
  • the reaction time is 20-40 min.
  • step S3 continue to repeat steps S2-S3 for at least N rounds, where N ⁇ 1 and N is an integer; or,
  • step S3 continue to repeat steps S2-S3 for at least N' rounds, N' ⁇ 0, N' is an integer, and only repeat step S2 in the last round.
  • the present invention provides a method for detecting a target nucleic acid, comprising: using the aforementioned nucleic acid hybridization method to obtain a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer; and detecting the hybrid using a first detection probe and/or a second detection probe, wherein the first detection probe includes a sequence that pairs and binds to at least a portion of a region of the second primer that is not paired with the first primer; the second detection probe includes a sequence that pairs and binds to at least a portion of a region of the third primer that is not paired with the second primer; and the first detection probe and the second detection probe contain markers that can detect signals.
  • the first detection probe includes a sequence that pairs with at least a portion of the second region in the second primer; the first detection probe includes a sequence that pairs with at least a portion of the seventh fragment in the second primer. A sequence that pairs and binds with at least a portion of the eighth segment.
  • the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer; the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.
  • the method further comprises:
  • step S2 detecting the hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe;
  • steps S2-S3 are repeated for at least N rounds after step S3, the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer is detected with the second detection probe, and optionally, auxiliary detection is performed using the first detection probe, where N is a natural number; or
  • step S3 continue to repeat steps S2-S3 for at least N' rounds, and in the last round, only repeat step S2, then use the first detection probe to detect the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer, and optionally, use the second detection probe for auxiliary detection, wherein N' is a positive integer.
  • the lengths of the first and second detection probes are independently 13-23 nt.
  • the present invention also provides a method for combined detection of nucleic acids and proteins, comprising:
  • the protein fluorescent staining comprises multiple rounds of protein staining using antibodies.
  • the biological sample containing nucleic acid is preferably fixed on a solid support.
  • the biological sample is a tissue/cell section sample.
  • the present invention provides a nucleic acid detection primer combination, which includes:
  • a first primer comprising: a. two pairing regions complementary to the target nucleic acid sequence to be detected; and b. a non-pairing region non-complementary to the target nucleic acid sequence to be detected, wherein the two pairing regions in a. are located at both ends of the first primer, and the non-pairing region comprises two groups of identical sequences, the two groups of identical sequences are respectively connected to the pairing regions at both ends of the first primer, and the two groups of identical sequences are connected by a first spacer sequence;
  • a second primer comprising: c. a pairing region complementary to any one of the two identical sequences in the non-pairing region of the first primer; and d. a non-pairing region non-complementary to the first primer, wherein the pairing region in c. comprises two sequences and is located at both ends of the second primer, respectively; and the non-pairing region in d. comprises two identical sequences and is connected to the pairing regions at both ends of the second primer, respectively, and the two identical sequences are connected by a second spacer sequence;
  • a third primer comprising: e. a pairing region complementary to any one of the two identical sequences in the non-pairing region of the second primer; and f. a non-pairing region non-complementary to the second primer, wherein the pairing region in e. comprises two sequences and is located at both ends of the third primer, respectively; the non-pairing region in f. comprises two identical sequences and is connected to the pairing regions at both ends of the third primer, respectively; and the two identical sequences are connected by a third spacer sequence.
  • the two groups of identical sequences in the non-paired region in b. are identical to the two groups of identical sequences in the non-paired region in f.
  • the lengths of the two pairing regions in a. are independently 15-20 nt.
  • the lengths of the two pairing regions in a. are 15 nt and 16 nt respectively. "Independent of each other" means that the lengths do not affect each other.
  • the lengths of the two pairing regions can be the same or different.
  • multiple target sites can be set according to the sequence length of the gene, for example, 10-30 sites can be set to achieve the binding of multiple first primers on the same target nucleic acid, that is, to achieve signal amplification of multiple sites.
  • the structure is as shown in Figure 1, the first segment and the second segment of the #1 primer (the first primer) (the two segments constitute the fourth (IV) region) bind to the mRNA on the glass slide to form a primary hybridization signal; and in addition to containing the first segment (marked with the number "1" in the figure, the other segments are represented in the same way, only the numbers are different) and the second segment, the #1 primer also contains two groups of respectively connected third segments and fourth segments (the two segments constitute the first (I) region), and they respectively bind to the fifth and sixth segments (the second and third segments) corresponding to the two #2 primers.
  • the fifth (V) region is formed by complementary binding; each #2 primer contains not only the fifth and sixth segments, but also two sets of connected seventh and eighth segments (the two together form the second (II) region), which complement each other with the corresponding ninth and tenth segments of the two #3 primers.
  • the #3 primer also contains two sets of sequences identical to the third and fourth segments of the #1 primer.
  • the two sets of connected third and fourth segments contained in the #3 primer can be used to complementarily bind with the fifth and sixth segments of the second primer in the next round.
  • the present invention can form an exponential amplification state through the complementary binding of multiple #2 primers and multiple #3 primers.
  • the capture of fluorescent signals can be achieved by targeting the approximately 15bp reading sequence (fluorescent probe) of the #2 or #3 primer with specific sequence patterns, making it easier and faster to detect the target nucleic acid.
  • the role of T4 DNA ligase is to connect the head and tail ends of this sequence to form a loop at the end of each amplification stage.
  • This type of primer sequence design can ensure targeted binding between primer sequences at each level, making capture easier; the setting of the double binding region facilitates further amplification, forming an exponential amplification effect; at the same time, the use of T4 DNA ligase instead of click chemistry to form a ring greatly reduces costs.
  • the first fragment and the adjacent third fragment, the second fragment and the adjacent fourth fragment, the fifth fragment and the adjacent seventh fragment, the sixth fragment and the adjacent eighth fragment, the ninth fragment and the adjacent third fragment, and the tenth fragment and the adjacent fourth fragment can each independently have a spacer sequence or be directly connected.
  • the length of the spacer sequence is preferably 2-5nt.
  • the target nucleic acid to be detected is single-stranded DNA or RNA.
  • the RNA can be mRNA directly transcribed in cells in a tissue section, and the single-stranded DNA can be single-stranded DNA derived from some tumor cells.
  • nucleic acid primer combination and kit provided by the present invention can be used for the following purposes:
  • RNA nucleic acids
  • tissue maps can be constructed, cell distribution within tissues can be determined, and biomarkers can be screened.
  • the nucleic acid primer combinations and kits of the present invention can also be used for digital medical imaging.
  • a method for in situ hybridization of a target nucleic acid comprising using the aforementioned nucleic acid detection primer combination to complementarily pair with a target nucleic acid and amplify the target nucleic acid signal, and detecting the target nucleic acid using a fluorescently labeled probe complementary to an unpaired region of the second or third primer.
  • the target nucleic acid is single-stranded DNA or RNA, and the fluorescently labeled probe has a probe length of 13-23 nt.
  • the present invention provides a fluorescence in situ hybridization method, which comprises the following steps:
  • step 6) using a ligase to link the third primer in step 6) into a ring, removing excess third primer not bound to the second primer, thereby obtaining a tissue section to be tested containing target RNA-circularized first primer-circularized second primer-circularized third primer;
  • the primers and the tissue to be tested are contacted in a hybridization reaction solution containing ethylene carbonate.
  • concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v/v.
  • the addition of ethylene carbonate to the buffer further shortens the incubation time between #1 and the target mRNA.
  • the present invention provides a method for joint detection of RNA and protein in tissue sections, comprising the following steps:
  • the tissue protein fluorescent staining includes multiple rounds of protein staining using antibodies.
  • the nucleic acid in situ hybridization method provided by the present invention is efficient, time-saving, inexpensive, and capable of combined RNA and protein detection. RNA fluorescence detection can amplify the signal without the need for excessive use of fluorescent probes, thus shortening the incubation time.
  • the nucleic acid in situ hybridization method provided by the present invention uses a primer combination that only requires changing the sequence of the first primer, while the second and third primers are universal primers, and the same fluorescent probe can be used for detection of different RNA targets, greatly reducing costs. After RNA detection, protein staining can be performed directly, and repeated positioning can be used to achieve combined detection.
  • RNA in situ hybridization was performed on mouse tumor tissue, which was an MDA-MB231 tumor model.
  • the paraffin blocks were purchased from Wuhan Saiweier.
  • reaction primers #1, #2, and #3 probe primers are all diluted to 100 ⁇ M using TE
  • read primer Dissolve each primer in varying amounts of TE buffer until completely dissolved. Then, dilute each primer in an EP tube at a 1:100 concentration (or the desired concentration, determined by calculation) and label it as the diluted primer tube.
  • Formal reaction Open the hot plate and adjust the temperature to 37°C. Place the creased sealing film in the center of the hot plate and seal it. Add 1 ml of regular washing buffer to the middle of the membrane, place the tissue section upside down in the center of the sealing membrane, and start the reaction timer;
  • primers used for RNA fluorescence in situ hybridization are shown in Table 1 below. Multiple primer combinations #1-1 to #1-16 were designed using CD8 mRNA as the detection target. Primers #2 and #3 were used for signal amplification, and read probes #2 and #3 were used for signal detection:
  • RNA fluorescence in situ hybridization detection The results of RNA fluorescence in situ hybridization detection are shown in FIG2 , indicating that the use of the primer combination #1-#3 of the present invention can greatly shorten the incubation time for fluorescence reading and detecting RNA hybridization signals, and can quickly detect the in situ expression of target RNA.
  • the inventors conducted a comparative experiment on the reaction time of primer #1 and the probe concentration in the reading buffer. As shown in Figure 2, the three experimental groups were divided into: 90-minute incubation time of the primary probe + 10 nM detection probe; 90-minute incubation time of the primary probe + 20 nM detection probe; and 150-minute incubation time of the primary probe + 20 nM detection probe. All other conditions of the three experimental schemes were the same. The results showed that the optimal reading probe concentration was 20 nM and the incubation time of the #1 reaction system was 90 minutes.
  • RNA in situ hybridization and tissue protein fluorescence staining were performed on mouse tumor tissue.
  • the tumor tissue was an MDA-MB231 tumor model, and the paraffin blocks were purchased from Wuhan Saiweier.
  • RNA fluorescence in situ hybridization detection The operation steps are the same as those in "2. RNA fluorescence in situ hybridization detection" of Example 1, wherein the reaction time in step f is 90 minutes.
  • antibody elution buffer (0.8% Beta-ME, 62.5 mM Tris-HCl, 1% SDS) (56°C, 10 minutes);
  • the reference terms “one embodiment”, “some embodiments”, “example”, “specific example”, “some implementation plans” or “some examples” mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention.
  • the schematic expressions of the above terms do not necessarily refer to the same embodiment or example.
  • the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
  • those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

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Abstract

Provided are a nucleic acid detection primer combination or kit, a nucleic acid hybridization method, a method for detecting a target nucleic acid, and a method for joint detection of a nucleic acid and a protein. By using the primer combination and the detection method, the incubation time for fluorescence reading/detecting a hybridization signal for a nucleic acid (such as RNA) can be shortened, ensuring that after hybridization of a nucleic acid molecular signal, multiple rounds of protein staining can still be carried out on a sample, i.e. an RNA-protein joint detection method, which provides two types of evidence for staining a target site by means of detection results.

Description

核酸检测方法及其应用Nucleic acid detection methods and their applications 技术领域Technical Field

本发明涉及核酸杂交技术领域，具体涉及核酸检测方法，及其与蛋白质检测的联合检测方法。The present invention relates to the field of nucleic acid hybridization technology, and in particular to a nucleic acid detection method and a combined detection method thereof with protein detection.

背景技术Background Art

荧光数字病理显微技术是生命科学领域的关键技术之一。目前应用最先进的组学方法结合荧光显微镜以及数字化各项参数，可以实现生物切片样本(例如组织切片样本)以百万像素的数字形式，呈现切片样本的以万计的诸多特征。Fluorescence digital pathology microscopy is a key technology in the life sciences. By combining state-of-the-art omics methods with fluorescence microscopy and digitized parameters, biological samples (such as tissue sections) can be visualized digitally in millions of pixels, displaying tens of thousands of features.

目前已研发一种快速多重的数字病理免疫荧光仪器，其研发基于桌面系列测序仪，已实现除了基础测序功能外，兼容组织蛋白质荧光染色与成像。目前此仪器着眼于组织切片蛋白质多轮染色、拍照功能，但是蛋白质多轮染色在潜在的未来数字病理诊断应用场景上，仅可被视为单一诊断依据，其仍然有较大提升空间。因此，在蛋白质多轮染色前提下增加目标靶点的RNA分子杂交信号捕捉与识别，能够进一步提升数字病理诊断的准确度。A rapid, multiplexed digital pathology immunofluorescence instrument has been developed. Based on a desktop series of sequencers, it now supports tissue protein fluorescence staining and imaging, in addition to basic sequencing capabilities. Currently, this instrument focuses on multiple rounds of protein staining and photography on tissue sections. However, in potential future digital pathology diagnostic applications, multiple rounds of protein staining can only be considered a single diagnostic basis, leaving significant room for improvement. Therefore, adding the capture and identification of RNA hybridization signals at the target site to multiple rounds of protein staining can further enhance the accuracy of digital pathology diagnoses.

然而，现有多重免疫荧光技术在需要在常温下对样品进行封闭，而且每一轮抗体洗脱后均需要重新进行封闭，增加了染色时间。传统免疫荧光染色时间较长，约210分钟，且传统染色方法缺少对单一切片的多重免疫荧光检测方法。迭代间接免疫荧光成像方法则为传统方法的改进版。在成像后加入抗体洗脱液(含有TCEP、尿素、盐酸胍、Tris-HCl)进行30分钟的抗体洗脱后重复染拍步骤。然而该抗体洗脱液粘度过高，容易造成组织脱落，破坏严重。且现有技术因信噪比问题，需进行长时间染色降低信噪比，才能达到预期效果。However, the existing multiple immunofluorescence technology needs to block the sample at room temperature, and each round of antibody elution needs to be re-blocked, which increases the staining time. Traditional immunofluorescence staining takes a long time, about 210 minutes, and the traditional staining method lacks a multiple immunofluorescence detection method for a single slice. The iterative indirect immunofluorescence imaging method is an improved version of the traditional method. After imaging, an antibody eluent (containing TCEP, urea, guanidine hydrochloride, Tris-HCl) is added for 30 minutes of antibody elution and then the staining step is repeated. However, the viscosity of the antibody eluent is too high, which can easily cause tissue shedding and serious damage. And due to the signal-to-noise ratio problem, the existing technology needs to be stained for a long time to reduce the signal-to-noise ratio in order to achieve the desired effect.

因此亟需开发一种能够大幅度缩短荧光读取/检测RNA杂交信号的孵育时间，且保证在RNA分子信号杂交之后，洗脱RNA探针荧光物质，继续在组织上实现蛋白质多轮染色的方法，即RNA-蛋白质联合检测方法，以通过检测结果给目标靶点染色提供双重证据。Therefore, there is an urgent need to develop a method that can significantly shorten the incubation time for fluorescent reading/detection of RNA hybridization signals, and ensure that after the RNA molecular signal hybridization, the RNA probe fluorescent substance is eluted to continue to achieve multiple rounds of protein staining on the tissue, that is, the RNA-protein combined detection method, so as to provide double evidence for the staining of the target through the detection results.

发明内容Summary of the Invention

本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提供核酸检测引物组合或套装，核酸杂交方法，检测靶标核酸的方法，以及核酸、蛋白质联合检测(联检)的方法等。利用本发明的引物组合以及检测方法，能够大大缩短荧光读取/检测核酸(如RNA)杂交信号的孵育时间，且保证在核酸分子信号杂交之后，继续在样本(例如组织)上实现蛋白质多轮染色，即核酸(如RNA)-蛋白质联合检测方法，通过检测结果能够给目标靶点染色提供核酸水平和蛋白水平的双重证据。The present invention is intended to solve one of the technical problems in the related art at least to a certain extent.For this reason, an object of the present invention is to provide a nucleic acid detection primer combination or suit, a nucleic acid hybridization method, a method for detecting a target nucleic acid, and a method for nucleic acid, protein joint detection (joint detection) etc. Utilizing the primer combination and detection method of the present invention, the incubation time of fluorescence reading/detection nucleic acid (such as RNA) hybridization signal can be greatly shortened, and it is ensured that after the nucleic acid molecule signal hybridization, multiple rounds of protein staining are continuously realized on the sample (such as tissue), i.e., a nucleic acid (such as RNA)-protein joint detection method, which can provide dual evidence of nucleic acid level and protein level to the target target staining by the detection result.

为此，本发明第一方面提供一种引物组合，所述引物组合包括：To this end, the first aspect of the present invention provides a primer combination, the primer combination comprising:

(1)一种或多种第一引物，其中，所述第一引物包括：与靶标核酸的片段配对结合的第四区域，以及至少一个与所述靶标核酸不配对结合的第一区域；(1) one or more first primers, wherein the first primers include: a fourth region that pairs and binds to a fragment of a target nucleic acid, and at least one first region that does not pair and bind to the target nucleic acid;

(2)第二引物，所述第二引物包括：与所述第一区域配对结合的第五区域，以及至少两个不与所述靶标核酸配对结合的第二区域；(2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid;

(3)第三引物，所述第三引物包括：与所述第二区域配对结合的第六区域，以及至少两个不与所述靶标核酸配对结合的第三区域。(3) A third primer, comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid.

本发明通过所述三级引物组合，保证三级引物之间有互补结合的结合域；且可在连接酶的作用下，保证每个层级的引物序列及时成环，进一步使三级引物紧密结合、指数形式扩增；这样独特的引物序列设计，根据三级引物杂交逻辑定向捕捉杂交信号，大幅度缩短了荧光读取/检测目标核酸杂交信号的孵育时间。例如，传统荧光原位杂交技术第一杂交引物需要过夜潮湿环境37℃孵育条件，现仅需90min左右即可完成类似效果的高效反应。The present invention utilizes the described tertiary primer combination to ensure that the three primers have complementary binding domains. Furthermore, under the action of a ligase, the primer sequences at each level can be looped in a timely manner, further enabling tight binding and exponential amplification of the three primers. This unique primer sequence design, based on the tertiary primer hybridization logic, allows for the targeted capture of hybridization signals, significantly shortening the incubation time for fluorescent reading/detection of target nucleic acid hybridization signals. For example, whereas traditional fluorescence in situ hybridization techniques require overnight incubation at 37°C in a humid environment, a similarly efficient reaction can now be completed in only about 90 minutes.

利用本发明的引物组合，可以将其应用于原位杂交，洗脱探针荧光物质后，继续在组织上实现蛋白质多轮染色，给目标靶点染色提供双重证据。The primer combination of the present invention can be applied to in situ hybridization. After the probe fluorescent substance is eluted, multiple rounds of protein staining can be continued on the tissue to provide double evidence for the staining of the target point.

根据本发明的实施方案，所述第一引物包括：与靶标核酸的片段配对结合的第四区域， 以及至少两个与所述靶标核酸的序列不配对结合的第一区域。According to an embodiment of the present invention, the first primer includes: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that bind to the target nucleic acid sequence without pairing.

根据本发明的实施方案，所述第二区域不与所述第一区域配对结合。According to an embodiment of the present invention, the second region does not pair-bind with the first region.

根据本发明的实施方案，所述第三区域不与所述第二区域配对结合。According to an embodiment of the present invention, the third region does not pair-bind with the second region.

根据本发明的实施方案，所述第四区域包括第一片段和第二片段，所述第一片段和第二片段分别位于所述第一引物的两端，所述第一区域位于所述第一片段和第二片段之间；每一个所述第一区域至少包括第三片段和第四片段；所述第五区域包括第五片段和第六片段，所述第五片段和第六片段分别位于所述第二引物的两端，所述第二区域位于所述第五片段和第六片段之间；每一个所述第二区域至少包括第七片段和第八片段；所述第六区域包括第九片段和第十片段，所述第九片段和第十片段分别位于所述第三引物的两端，所述第三区域位于所述第九片段和第十片段之间；每一个所述第三区域至少包括所述第三片段和第四片段。According to an embodiment of the present invention, the fourth region includes a first fragment and a second fragment, the first fragment and the second fragment are respectively located at the two ends of the first primer, and the first region is located between the first fragment and the second fragment; each first region includes at least a third fragment and a fourth fragment; the fifth region includes a fifth fragment and a sixth fragment, the fifth fragment and the sixth fragment are respectively located at the two ends of the second primer, and the second region is located between the fifth fragment and the sixth fragment; each second region includes at least a seventh fragment and an eighth fragment; the sixth region includes a ninth fragment and a tenth fragment, the ninth fragment and the tenth fragment are respectively located at the two ends of the third primer, and the third region is located between the ninth fragment and the tenth fragment; each third region includes at least the third fragment and the fourth fragment.

根据本发明的实施方案，所述引物组合包括多种第一引物，其中每一种所述第一引物的第四区域与不同靶标核酸的片段或相同靶标核酸的不同片段配对结合。According to an embodiment of the present invention, the primer combination includes a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid.

根据本发明的实施方案，所述第一引物的5’端的第一个碱基与3’端的最后一个碱基对应与其配对结合的所述靶标核酸的片段上相邻的碱基。According to an embodiment of the present invention, the first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the fragment of the target nucleic acid to which they are paired and bound.

根据本发明的实施方案，所述第一片段和第二片段的长度各自独立地为15-20nt。According to an embodiment of the present invention, the lengths of the first fragment and the second fragment are each independently 15-20 nt.

根据本发明的实施方案，所述第三片段和第四片段的长度各自独立地为8-20nt，优选10-16nt。According to an embodiment of the present invention, the lengths of the third fragment and the fourth fragment are each independently 8-20 nt, preferably 10-16 nt.

根据本发明的实施方案，所述第五片段和第六片段的长度各自独立地为8-20nt，优选10-16nt。According to an embodiment of the present invention, the lengths of the fifth fragment and the sixth fragment are each independently 8-20 nt, preferably 10-16 nt.

根据本发明的实施方案，所述第七片段和第八片段的长度各自独立地为8-20nt，优选10-16nt。According to an embodiment of the present invention, the lengths of the seventh fragment and the eighth fragment are each independently 8-20 nt, preferably 10-16 nt.

根据本发明的实施方案，所述第九片段和第十片段的长度各自独立地为8-20nt，优选10-16nt。According to an embodiment of the present invention, the lengths of the ninth fragment and the tenth fragment are each independently 8-20 nt, preferably 10-16 nt.

根据本发明的实施方案，所述第一引物包括一个以上的所述第一区域，各个第一区域之间通过第一间隔序列相连。According to an embodiment of the present invention, the first primer includes more than one first region, and the first regions are connected by a first spacer sequence.

根据本发明的实施方案，在第二引物中，各个所述第二区域之间通过第二间隔序列相连。According to an embodiment of the present invention, in the second primer, each of the second regions is connected by a second spacer sequence.

根据本发明的实施方案，在第三引物中，各个所述第三区域之间通过第三间隔序列相连。According to an embodiment of the present invention, in the third primer, each of the third regions is connected by a third spacer sequence.

根据本发明的实施方案，所述第一间隔序列长度至少为1nt，优选1-30nt，进一步优选2-5nt。According to an embodiment of the present invention, the length of the first spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.

根据本发明的实施方案，所述第二间隔序列长度至少为1nt，优选1-30nt，进一步优选2-5nt。According to an embodiment of the present invention, the length of the second spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.

根据本发明的实施方案，所述第三间隔序列长度至少为1nt，优选1-30nt，进一步优选2-5nt。According to an embodiment of the present invention, the length of the third spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.

本发明第二方面提供一种套装，所述套装包括：第一方面所述的引物组合，以及第一检测探针和/或第二检测探针；其中，所述第一检测探针包括与所述第二引物中的不与所述第一引物配对的区域的至少一部分配对结合的序列，所述第二检测探针包括与所述第三引物中的不与所述第二引物配对的区域的至少一部分配对结合的序列，所述第一检测探针和所述第二检测探针含有可检测信号的标记物。The second aspect of the present invention provides a kit, which comprises: the primer combination described in the first aspect, and a first detection probe and/or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of the region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.

根据本发明的实施方案，所述第一检测探针包括与所述第二引物中的第二区域的至少一部分配对结合的序列。According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.

根据本发明的实施方案，所述第一检测探针包括与所述第二引物中的第七片段的至少一部分和第八片段的至少一部分配对结合的序列。According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.

根据本发明的实施方案，所述第二检测探针包括与所述第三引物中的第三区域的至少一部分配对结合的序列。According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.

根据本发明的实施方案，所述第二检测探针包括与所述第三引物中的第三片段的至少一部分和第四片段的至少一部分配对结合的序列。According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

根据本发明的实施方案，所述标记物包括放射性标记物或非放射性标记物。 According to an embodiment of the present invention, the label comprises a radioactive label or a non-radioactive label.

根据本发明的实施方案，所述非放射性标记物包括荧光基团。According to an embodiment of the present invention, the non-radioactive label comprises a fluorescent group.

本发明第三方面提供一种核酸杂交方法，所述方法包括以下步骤：A third aspect of the present invention provides a nucleic acid hybridization method, comprising the following steps:

S1.将含有靶标核酸的生物样本与第一方面所述的引物组合中的一种或多种第一引物接触，所述一种或多种第一引物中的第四区域与靶标核酸的片段配对结合，获得靶标核酸-第一引物杂交体1；S1. contacting a biological sample containing a target nucleic acid with one or more first primers in the primer combination described in the first aspect, wherein the fourth region of the one or more first primers pairs and binds to a fragment of the target nucleic acid to obtain a target nucleic acid-first primer hybrid 1;

S2.将步骤S1获得的产物与第一方面所述的引物组合中的第二引物接触，所述第二引物的第五区域与所述第一引物的第一区域配对结合，获得靶标核酸-第一引物-第二引物杂交体2；S2. contacting the product obtained in step S1 with the second primer in the primer combination described in the first aspect, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain a target nucleic acid-first primer-second primer hybrid 2;

任选地S3.将步骤S2获得的产物与第一方面所述的引物组合中的第三引物接触，所述第三引物的第六区域与所述第二引物的第二区域配对结合，获得靶标核酸-第一引物-第二引物-第三引物杂交体3。Optionally, S3. the product obtained in step S2 is contacted with the third primer in the primer combination described in the first aspect, and the sixth region of the third primer is paired and bound with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3.

根据本发明的实施方案，所述方法进一步包括：步骤S1-S3中的接触是在含有碳酸乙烯酯的杂交反应液中进行的，所述杂交反应液中碳酸乙烯酯的浓度为5-40％v/v。According to an embodiment of the present invention, the method further comprises: the contacting in steps S1 to S3 is performed in a hybridization reaction solution containing ethylene carbonate, and the concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v/v.

根据本发明的实施方案，所述杂交反应液进一步包括硫酸葡聚糖和Triton-X-100，其中，所述硫酸葡聚糖的浓度为5-20％，所述Triton-X-100的浓度为0.05-0.5％。According to an embodiment of the present invention, the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

根据本发明的实施方案，所述方法进一步包括：在进行步骤S2之前，利用连接酶将步骤S1反应结束后体系中存在的第一引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合靶标核酸的第一引物。According to an embodiment of the present invention, the method further includes: before performing step S2, using a ligase to connect the first primer present in the system after the reaction of step S1 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess first primers that are not bound to the target nucleic acid.

根据本发明的实施方案，所述方法进一步包括：在进行步骤S3之前，利用连接酶将步骤S2反应结束后体系中存在的第二引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合第一引物的第二引物。According to an embodiment of the present invention, the method further includes: before performing step S3, using a ligase to connect the second primer present in the system after the reaction of step S2 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess second primers that are not bound to the first primer.

根据本发明的实施方案，所述方法进一步包括：在进行步骤S3之后，利用连接酶将步骤S3反应结束后体系中存在的第三引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合第二引物的第三引物。According to an embodiment of the present invention, the method further includes: after performing step S3, using a ligase to connect the third primer present in the system after the reaction of step S3 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess third primers that are not bound to the second primer.

根据本发明的实施方案，所述方法进一步包括：利用所述连接酶进行连接反应的反应温度为30-40℃，反应时间为5-30min。According to an embodiment of the present invention, the method further comprises: performing a ligation reaction using the ligase at a reaction temperature of 30-40° C. and a reaction time of 5-30 min.

根据本发明的实施方案，所述连接酶为T4连接酶。According to an embodiment of the present invention, the ligase is T4 ligase.

根据本发明的实施方案，所述清洗液包括碳酸乙烯酯，所述碳酸乙烯酯在所述清洗液中的浓度为5-40％v/v。According to an embodiment of the present invention, the cleaning solution comprises ethylene carbonate, and the concentration of ethylene carbonate in the cleaning solution is 5-40% v/v.

根据本发明的实施方案，所述清洗液进一步包括硫酸葡聚糖和Triton-X-100，其中，所述硫酸葡聚糖的浓度为5-20％，所述Triton-X-100的浓度为0.05-0.5％。According to an embodiment of the present invention, the cleaning solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

根据本发明的实施方案，步骤S1中配对结合的反应温度为30-40℃，反应时间为30-120min。According to an embodiment of the present invention, the reaction temperature for pairing and binding in step S1 is 30-40° C., and the reaction time is 30-120 min.

根据本发明的实施方案，步骤S2中配对结合的反应温度为30-40℃，反应时间为20-40min。According to an embodiment of the present invention, the reaction temperature for the pairing binding in step S2 is 30-40° C., and the reaction time is 20-40 min.

根据本发明的实施方案，步骤S3中配对结合的反应温度为30-40℃，反应时间为20-40min。According to an embodiment of the present invention, the reaction temperature for the pairing binding in step S3 is 30-40° C., and the reaction time is 20-40 min.

根据本发明的实施方案，所述方法进一步包括：According to an embodiment of the present invention, the method further comprises:

Ⅰ.在步骤S3之后，继续重复步骤S2-S3至少N轮，N≥1，N为整数；或者，Ⅰ. After step S3, continue to repeat steps S2-S3 for at least N rounds, where N≥1 and N is an integer; or,

Ⅱ.在步骤S3之后，继续重复步骤S2-S3至少N’轮，N’≥0，N’为整数，且在最后一轮仅重复步骤S2。II. After step S3, continue to repeat steps S2-S3 for at least N' rounds, N'≥0, N' is an integer, and only repeat step S2 in the last round.

本发明第四方面提供一种检测靶标核酸的方法，所述方法包括：利用第三方面所述的核酸杂交方法，获得靶标核酸、第一引物、第二引物、任选的第三引物组成的杂交体，利用第一检测探针和/或第二检测探针检测所述杂交体；其中，所述第一检测探针包括与所述第二引物中的不与所述第一引物配对的区域的至少一部分配对结合的序列，所述第二检测探针包括与所述第三引物中的不与所述第二引物配对的区域的至少一部分配对结合的序列，所述第一检测探针和所述第二检测探针含有可检测信号的标记物。The fourth aspect of the present invention provides a method for detecting a target nucleic acid, the method comprising: using the nucleic acid hybridization method described in the third aspect to obtain a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer, and detecting the hybrid using a first detection probe and/or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of a region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of a region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.

根据本发明的实施方案，所述第一检测探针包括与所述第二引物中的第二区域的至少一部分配对结合的序列。According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.

根据本发明的实施方案，所述第一检测探针包括与所述第二引物中的第七片段的至少一 部分和第八片段的至少一部分配对结合的序列。According to an embodiment of the present invention, the first detection probe comprises at least one of the seventh fragments in the second primer. The sequence of the first portion and the eighth segment is paired and bound to at least a portion of the first portion.

根据本发明的实施方案，所述第二检测探针包括与所述第三引物中的第三区域的至少一部分配对结合的序列。According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.

根据本发明的实施方案，所述第二检测探针包括与所述第三引物中的第三片段的至少一部分和第四片段的至少一部分配对结合的序列。根据本发明的实施方案，所述靶标核酸为单链DNA或RNA。According to an embodiment of the present invention, the second detection probe comprises a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer. According to an embodiment of the present invention, the target nucleic acid is single-stranded DNA or RNA.

根据本发明的实施方案，所述方法进一步包括：在步骤S2之后，以所述第一检测探针检测所述靶标核酸、第一引物以及第二引物组成的杂交体。According to an embodiment of the present invention, the method further comprises: after step S2, detecting a hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe.

根据本发明的实施方案，所述方法进一步包括：在步骤S3之后继续重复步骤S2-S3至少N轮，以所述第二检测探针检测所述靶标核酸、第一引物、第二引物以及第三引物组成的杂交体，任选地，利用所述第一检测探针进行辅助检测，其中N为自然数。According to an embodiment of the present invention, the method further includes: after step S3, continuing to repeat steps S2-S3 for at least N rounds, detecting the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer with the second detection probe, and optionally, using the first detection probe for auxiliary detection, where N is a natural number.

根据本发明的另一个实施方案，在步骤S3之后继续重复步骤S2-S3至少N’轮，且在最后一轮仅重复步骤S2的情况，则以所述第一检测探针检测所述靶标核酸、第一引物、第二引物以及第三引物组成的杂交体，任选地，利用所述第二检测探针进行辅助检测，其中N’为正整数。According to another embodiment of the present invention, after step S3, steps S2-S3 are repeated for at least N' rounds, and in the last round, only step S2 is repeated. Then, the first detection probe is used to detect the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer, and optionally, the second detection probe is used for auxiliary detection, wherein N' is a positive integer.

根据本发明的实施方案，所述第一、二检测探针的探针长度分别独立地为13-23nt。According to an embodiment of the present invention, the lengths of the first and second detection probes are independently 13-23 nt.

本发明第五方面提供一种核酸、蛋白质联合检测的方法，所述方法包括以下步骤：A fifth aspect of the present invention provides a method for combined detection of nucleic acids and proteins, comprising the following steps:

1)利用第四方面所述的检测靶标核酸的方法对含有核酸的待测生物样本进行核酸检测；以及1) performing nucleic acid detection on a biological sample containing nucleic acid using the method for detecting target nucleic acid described in the fourth aspect; and

2)对所述生物样本进行蛋白质荧光染色与成像。2) performing protein fluorescence staining and imaging on the biological sample.

根据本发明的实施方案，所述蛋白质荧光染色包括利用蛋白质标记物(例如抗体)进行蛋白质多轮染色。According to an embodiment of the present invention, the protein fluorescent staining comprises performing multiple rounds of protein staining using protein markers (eg, antibodies).

本发明第六方面提供第一方面所述的引物组合、第二方面所述的套装在以下中的用途：The sixth aspect of the present invention provides uses of the primer combination described in the first aspect and the kit described in the second aspect in the following:

a.核酸原位杂交；a. Nucleic acid in situ hybridization;

b.核酸、蛋白质联合检测；b. Combined detection of nucleic acid and protein;

c.组织图谱构建；c. Organizational map construction;

d.确定组织中细胞分布；d. Determine the distribution of cells in tissues;

e.数字病理成像和分析；e. Digital pathology imaging and analysis;

f.生物标志物的筛选和发现。f. Biomarker screening and discovery.

根据本发明的实施方案，在核酸、蛋白质联合检测中，待测生物样本优选地固定在固体支持物上。根据本发明的实施方案，所述待测生物样本为组织/细胞切片类样本。According to an embodiment of the present invention, in the combined detection of nucleic acids and proteins, the biological sample to be tested is preferably fixed on a solid support. According to an embodiment of the present invention, the biological sample to be tested is a tissue/cell section sample.

本发明是一种对于传统原位杂交染色方法的改进，此改进方法可大大提高染色效率，缩短染色时间。本发明也适用于普通传统手动染色系统，提高组织切片染色效率，降低操作人员的工作量，缩短工作时长。该方法具有广泛的潜在应用价值，包括组织图谱、RNA原位杂交、适配体染色等。可供动植物细胞分布，肿瘤和疾病图谱的构建，以及数字病理学和生物标志物的筛选和发现。This invention improves upon traditional in situ hybridization staining methods, significantly increasing staining efficiency and shortening staining time. It is also applicable to conventional manual staining systems, improving tissue section staining efficiency, reducing operator workload, and shortening working hours. This method has a wide range of potential applications, including tissue mapping, RNA in situ hybridization, and aptamer staining. It can be used to construct maps of plant and animal cell distribution, tumors, and diseases, as well as for digital pathology and biomarker screening and discovery.

发明人利用前面所述的引物组合进行荧光原位杂交，旨在实现快速多重的数字病理免疫荧光仪器中蛋白质多轮染色前提下，增加目标靶点的核酸分子杂交信号捕捉与识别，以实现核酸和蛋白质在机器上的联检。实验设计包括组织切片准备(脱蜡水化抗原修复)、核酸杂交分子序列设计与准备、核酸杂交过程试剂制备、蛋白质多轮染色试剂制备、一体化自动染拍整个过程。在原本蛋白质多轮染色之前，在快速多重的数字病理免疫荧光仪器的自动多轮染拍基础上，增加组织切片核酸分子多轮杂交、扩增、荧光信号读取、荧光信号洗脱环节，以此实现核酸、蛋白质联检的效果。The inventors used the primer combination described above to perform fluorescence in situ hybridization, aiming to increase the capture and identification of nucleic acid molecule hybridization signals of the target target under the premise of multiple rounds of protein staining in the fast and multiple digital pathology immunofluorescence instrument, so as to realize the joint detection of nucleic acids and proteins on the machine. The experimental design includes tissue section preparation (dewaxing and hydration antigen repair), nucleic acid hybridization molecule sequence design and preparation, nucleic acid hybridization process reagent preparation, protein multiple rounds of staining reagent preparation, and integrated automatic staining and shooting of the entire process. Before the original multiple rounds of protein staining, on the basis of the automatic multiple rounds of staining of the fast and multiple digital pathology immunofluorescence instrument, the multiple rounds of hybridization of nucleic acid molecules in tissue sections, amplification, fluorescence signal reading, and fluorescence signal elution are added to achieve the effect of joint detection of nucleic acids and proteins.

本发明提供的技术方案具有以下有益效果：The technical solution provided by the present invention has the following beneficial effects:

(1)相比传统荧光原位杂交技术，引物与靶标序列杂交需要过夜潮湿环境37℃孵育条件，本发明引物组合中第一引物(下文也称#1引物)仅需90min即可完成类似效果的高效反应；(1) Compared with traditional fluorescence in situ hybridization technology, which requires overnight incubation at 37°C in a humid environment for primers to hybridize with target sequences, the first primer in the primer combination of the present invention (hereinafter referred to as primer #1) can complete a similar efficient reaction in just 90 minutes;

(2)传统杂交技术，荧光探针需要对各个靶标进行定制，此方法只需改变#1杂交引物即 可，无需重新定制荧光探针，针对不同靶标序列，第二引物(下文也称#2引物)、第三引物(下文也称#3引物)是通用的，可大大降低成本；(2) In traditional hybridization technology, fluorescent probes need to be customized for each target. This method only requires changing the #1 hybridization primer. Yes, there is no need to customize the fluorescent probe. The second primer (hereinafter referred to as primer #2) and the third primer (hereinafter referred to as primer #3) are universal for different target sequences, which can greatly reduce costs.

(3)传统荧光原位杂交技术局限于RNA/DNA分子层面杂交信号捕捉，而不能在分子基础上增加目标基因蛋白质信号捕捉；(3) Traditional fluorescence in situ hybridization technology is limited to capturing hybridization signals at the RNA/DNA molecular level, and cannot increase the capture of target gene protein signals on a molecular basis;

(4)本发明中提供的引物组合应用于生物样本(例如组织切片)的核酸、蛋白质联合检测，核酸、蛋白质自动化染拍一体机，保证位置信息的可重复性以便背景矫正以及快速抗体孵育，本发明中的核酸、蛋白质联检可完全通过测序仪形成自动化染拍平台。(4) The primer combination provided in the present invention is applied to the joint detection of nucleic acids and proteins in biological samples (such as tissue sections). The nucleic acid and protein automated dyeing and shooting machine ensures the repeatability of position information for background correction and rapid antibody incubation. The nucleic acid and protein joint detection in the present invention can be completely formed into an automated dyeing and shooting platform through a sequencer.

本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

图1显示了利用第一引物、第二引物以及第三引物进行RNA荧光检测时，扩增过程的示意图；FIG1 is a schematic diagram showing the amplification process when performing RNA fluorescence detection using a first primer, a second primer, and a third primer;

图2显示了实施例1中靶标CD8 mRNA的荧光原位杂交在不同的反应条件下的对比结果，其中A.不同读取探针浓度下荧光原位杂交结果；B.不同读取探针浓度下荧光原位杂交统计结果；Figure 2 shows the comparative results of fluorescence in situ hybridization of the target CD8 mRNA under different reaction conditions in Example 1, wherein A. fluorescence in situ hybridization results under different read probe concentrations; B. statistical results of fluorescence in situ hybridization under different read probe concentrations;

图3显示了实施例2中靶标CD8 mRNA的荧光原位杂交在10％碳酸乙烯酯反应体系(左图)和10％聚乙烯胺反应体系(右图)的对比结果；Figure 3 shows the comparative results of fluorescence in situ hybridization of the target CD8 mRNA in Example 2 in a 10% ethylene carbonate reaction system (left) and a 10% polyvinylamine reaction system (right);

图4显示了实施例3中靶标蛋白PD-L1、CD8蛋白的组织蛋白质荧光染色结果。FIG4 shows the results of tissue protein fluorescence staining of the target proteins PD-L1 and CD8 protein in Example 3.

发明详细描述Detailed Description of the Invention

下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:APRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体：实验室手册》(ANTIBODIES,ALABORATORYMANUAL)，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。Conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., eds., (1987)); METHODS IN MOLECULAR BIOLOGY (eds. F.M. Ausubel et al., (1987)); IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames, and G.R. Taylor, eds. (1995)), ANTIBODIES: A LABORATORY MANUAL (Harlow and Lane, eds. (1988)), and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除 其他方面的内容。In this article, the term "include" or "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding Other content.

在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

在本文中，术语“引物”是指人工合成的寡核苷酸序列，其与靶区域的一条模板链的至少一部分互补配对，引物可以为单链的DNA引物或者RNA引物。Herein, the term "primer" refers to an artificially synthesized oligonucleotide sequence that is complementary to at least a portion of a template strand of a target region. The primer may be a single-stranded DNA primer or an RNA primer.

根据本发明一个具体的实施方案，本发明提供一种引物组合，包括：According to a specific embodiment of the present invention, the present invention provides a primer combination comprising:

(1)一种或多种第一引物，其中，所述第一引物包括：与靶标核酸的片段配对结合的第四区域，以及至少一个与所述靶标核酸不配对结合的第一区域；(1) one or more first primers, wherein the first primers include: a fourth region that pairs and binds to a fragment of a target nucleic acid, and at least one first region that does not pair and bind to the target nucleic acid;

(2)第二引物，所述第二引物包括：与所述第一区域配对结合的第五区域，以及至少两个不与所述靶标核酸配对结合的第二区域；(2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid;

(3)第三引物，所述第三引物包括：与所述第二区域配对结合的第六区域，以及至少两个不与所述靶标核酸配对结合的第三区域。(3) A third primer, comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid.

需要说明的是，本发明所述引物组合中的第一引物，可以设计为多种不同的第一引物序列，这些不同的序列可以分别与所述靶标核酸的不同区段互补配对，并基于第一引物与靶标核酸结合后，进一步与第二引物、第三引物杂交，形成多个靶标核酸-引物杂交体。It should be noted that the first primer in the primer combination of the present invention can be designed as a plurality of different first primer sequences, and these different sequences can be complementary paired with different segments of the target nucleic acid, respectively. After the first primer binds to the target nucleic acid, it further hybridizes with the second primer and the third primer to form multiple target nucleic acid-primer hybrids.

利用本发明的引物组合，可以将其应用于原位杂交，洗脱探针荧光物质后，继续在组织上实现蛋白质多轮染色，给目标靶点染色提供双重证据。The primer combination of the present invention can be applied to in situ hybridization. After the probe fluorescent substance is eluted, multiple rounds of protein staining can be continued on the tissue to provide double evidence for the staining of the target point.

根据本发明一个具体的实施方案，所述第一引物包括：与靶标核酸的片段配对结合的第四区域，以及至少两个与所述靶标核酸的序列不配对结合的第一区域。According to a specific embodiment of the present invention, the first primer includes: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that do not pair and bind to a sequence of the target nucleic acid.

根据本发明一个具体的实施方案，所述第二区域不与所述第一区域配对结合；所述第三区域不与所述第二区域配对结合。According to a specific embodiment of the present invention, the second region does not pair with the first region; and the third region does not pair with the second region.

根据本发明一个具体的实施方案，本发明的引物组合中，保证第二区域不与所述第一区域配对结合，这样的引物设计，能够保证第二引物的第五区域与第二区域不与第一引物的第一区域竞争性结合。According to a specific embodiment of the present invention, in the primer combination of the present invention, it is ensured that the second region does not pair and bind with the first region. Such primer design can ensure that the fifth region of the second primer and the second region do not competitively bind with the first region of the first primer.

根据本发明一个具体的实施方案，本发明的引物组合中，保证所述第三区域不与所述第二区域配对结合，这样的引物设计，能够保证第三引物的第三区域与第六区域不与第二引物的第二区域竞争性结合。According to a specific embodiment of the present invention, in the primer combination of the present invention, it is ensured that the third region does not pair and bind with the second region. Such primer design can ensure that the third region and the sixth region of the third primer do not competitively bind with the second region of the second primer.

根据本发明一个具体的实施方案，所述第四区域包括第一片段和第二片段，所述第一片段和第二片段分别位于所述第一引物的两端，所述第一区域位于所述第一片段和第二片段之间；每一个所述第一区域至少包括第三片段和第四片段；所述第五区域包括第五片段和第六片段，所述第五片段和第六片段分别位于所述第二引物的两端，所述第二区域位于所述第五片段和第六片段之间；每一个所述第二区域至少包括第七片段和第八片段；所述第六区域包括第九片段和第十片段，所述第九片段和第十片段分别位于所述第三引物的两端，所述第三区域位于所述第九片段和第十片段之间；每一个所述第三区域至少包括所述第三片段和第四片段。According to a specific embodiment of the present invention, the fourth region includes a first fragment and a second fragment, the first fragment and the second fragment are respectively located at the two ends of the first primer, and the first region is located between the first fragment and the second fragment; each first region includes at least a third fragment and a fourth fragment; the fifth region includes a fifth fragment and a sixth fragment, the fifth fragment and the sixth fragment are respectively located at the two ends of the second primer, and the second region is located between the fifth fragment and the sixth fragment; each second region includes at least a seventh fragment and an eighth fragment; the sixth region includes a ninth fragment and a tenth fragment, the ninth fragment and the tenth fragment are respectively located at the two ends of the third primer, and the third region is located between the ninth fragment and the tenth fragment; each third region includes at least the third fragment and the fourth fragment.

根据本发明一个具体的实施方案，所述引物组合包括多种第一引物，其中每一种所述第一引物的第四区域与不同靶标核酸的片段或相同靶标核酸的不同片段配对结合。According to a specific embodiment of the present invention, the primer combination includes a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid.

本发明提供的引物组合，当设计为包含：与不同靶标核酸的片段互补的第一片段和第二片段以及至少两个与所述靶标核酸的序列不配对结合的第一区域时，可以用于同时检测多个靶标核酸。根据本发明的实施方案，所述第一引物的5’端的第一个碱基与3’端的最后一个碱基对应与其配对结合的所述靶标核酸的片段上相邻的碱基。The primer combination provided by the present invention, when designed to include: a first segment and a second segment complementary to fragments of different target nucleic acids, and at least two first regions that do not bind to the sequence of the target nucleic acids, can be used to simultaneously detect multiple target nucleic acids. According to an embodiment of the present invention, the first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the target nucleic acid fragments to which they bind.

第一引物的5’端的第一个碱基与3’端的最后一个碱基对应与其配对结合的所述靶标核酸的片段上相邻的碱基是指第一引物与靶标核酸的片段配对结合时，第一引物的5’端和3’端分别与靶标核酸的片段配对结合，中间区段(第一区域)不结合，第一引物形成环状结构，第一引物的5’端的第一个碱基与3’端的最后一个碱基相邻，便于后续进行连接酶连接成环。The first base at the 5' end of the first primer and the last base at the 3' end correspond to adjacent bases on the target nucleic acid fragment with which they are paired and bound. This means that when the first primer is paired and bound to the target nucleic acid fragment, the 5' end and 3' end of the first primer are respectively paired and bound to the target nucleic acid fragment, and the middle segment (first region) is not bound. The first primer forms a ring structure, and the first base at the 5' end of the first primer is adjacent to the last base at the 3' end, which facilitates subsequent ligation into a ring by a ligase.

需要说明的是，本发明提及的“至少两个”是指两个以上、包括三个、四个、五个以及以上等，根据本发明一个具体的实施方案，本发明提供的引物组合，包括： It should be noted that the "at least two" mentioned in the present invention refers to more than two, including three, four, five and more. According to a specific embodiment of the present invention, the primer combination provided by the present invention includes:

(1)一种或多种第一引物，其中，所述第一引物包括：与靶标核酸的片段配对结合的第四区域，以及两个与所述靶标核酸不配对结合的第一区域；(1) one or more first primers, wherein the first primers include: a fourth region that binds to a target nucleic acid fragment, and two first regions that do not bind to the target nucleic acid;

(2)第二引物，所述第二引物包括：与所述第一区域配对结合的第五区域，以及两个不与所述靶标核酸配对结合的第二区域；(2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and two second regions that do not pair and bind to the target nucleic acid;

(3)第三引物，所述第三引物包括：与所述第二区域配对结合的第六区域，以及两个不与所述靶标核酸配对结合的第三区域。(3) A third primer, comprising: a sixth region that pairs and binds to the second region, and two third regions that do not pair and bind to the target nucleic acid.

根据本发明的实施方案，所述第一片段和第二片段的长度各自独立地为15-20nt。例如，第一片段和第二片段的长度各自独立地为15nt、16nt、17nt、18nt、19nt、20nt。According to an embodiment of the present invention, the length of the first fragment and the second fragment are each independently 15-20 nt. For example, the length of the first fragment and the second fragment are each independently 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt.

根据本发明的实施方案，所述第三片段、第四片段、第五片段、第六片段、第七片段、第八片段、第九片段和第十片段的长度各自独立地为8-20nt，优选10-16nt。例如，所述第三片段、第四片段、第五片段、第六片段、第七片段、第八片段、第九片段和第十片段的长度各自独立地为10nt、11nt、12nt、13nt、14nt、15nt或16nt。According to an embodiment of the present invention, the length of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment is each independently 8-20 nt, preferably 10-16 nt. For example, the length of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment is each independently 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt or 16 nt.

根据本发明的实施方案，所述第一引物包括一个以上的所述第一区域，各个第一区域之间通过第一间隔序列相连。所述第一间隔序列的设置使得第二引物更容易结合到第一引物上。According to an embodiment of the present invention, the first primer includes more than one first region, and the first regions are connected by a first spacer sequence. The provision of the first spacer sequence makes it easier for the second primer to bind to the first primer.

根据本发明的实施方案，在第二引物中，各个所述第二区域之间通过第二间隔序列相连。所述第二间隔序列的设置使得第三引物更容易结合到第二引物上。According to an embodiment of the present invention, in the second primer, each of the second regions is connected by a second spacer sequence. The provision of the second spacer sequence makes it easier for the third primer to bind to the second primer.

根据本发明的实施方案，在第三引物中，各个所述第三区域之间通过第三间隔序列相连。所述第三间隔序列的设置使得下一轮第二引物更容易结合到第三引物上。According to an embodiment of the present invention, in the third primer, each of the third regions is connected by a third spacer sequence. The provision of the third spacer sequence makes it easier for the second primer in the next round to bind to the third primer.

根据本发明的实施方案，所述第一间隔序列、所述第二间隔序列和所述第三间隔序列的长度独立地至少为1nt，优选1-30nt，进一步优选2-5nt。例如，所述第一间隔序列、所述第二间隔序列和所述第三间隔序列的长度各自独立地为2nt、3nt、4nt或5nt。According to an embodiment of the present invention, the length of the first spacer sequence, the second spacer sequence and the third spacer sequence is independently at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt. For example, the length of the first spacer sequence, the second spacer sequence and the third spacer sequence is each independently 2 nt, 3 nt, 4 nt or 5 nt.

本发明还提供一种套装，其包括：前面所述的引物组合，以及第一检测探针和/或第二检测探针；其中，所述第一检测探针包括与所述第二引物中的不与所述第一引物配对的区域的至少一部分配对结合的序列，所述第二检测探针包括与所述第三引物中的不与所述第二引物配对的区域的至少一部分配对结合的序列，所述第一检测探针和所述第二检测探针含有可检测信号的标记物。The present invention also provides a kit comprising: the primer combination described above, and a first detection probe and/or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of the region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.

根据本发明的实施方案，所述第一检测探针包括与所述第二引物中的第二区域的至少一部分配对结合的序列。According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.

根据本发明的实施方案，所述第一检测探针包括与所述第二引物中的第七片段的至少一部分和第八片段的至少一部分配对结合的序列。According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.

根据本发明的实施方案，所述第二检测探针包括与所述第三引物中的第三区域的至少一部分配对结合的序列。According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.

根据本发明的实施方案，所述第二检测探针包括与所述第三引物中的第三片段的至少一部分和第四片段的至少一部分配对结合的序列。According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

根据本发明的实施方案，所述标记物包括放射性标记物或非放射性标记物。根据本发明的实施方案，所述非放射性标记物包括荧光基团。According to an embodiment of the present invention, the label comprises a radioactive label or a non-radioactive label. According to an embodiment of the present invention, the non-radioactive label comprises a fluorescent group.

本发明还提供一种核酸杂交方法，所述方法包括：The present invention also provides a nucleic acid hybridization method, comprising:

S1.将含有靶标核酸的生物样本与前面所述的引物组合中的一种或多种第一引物接触，所述一种或多种第一引物中的第四区域与靶标核酸的片段配对结合，获得靶标核酸-第一引物杂交体1；S1. contacting a biological sample containing a target nucleic acid with one or more first primers from the primer combination described above, wherein the fourth region of the one or more first primers pairs and binds to a fragment of the target nucleic acid to obtain a target nucleic acid-first primer hybrid 1;

S2.将步骤S1获得的产物与前面所述的引物组合中的第二引物接触，所述第二引物的第五区域与所述第一引物的第一区域配对结合，获得靶标核酸-第一引物-第二引物杂交体2；S2. contacting the product obtained in step S1 with the second primer in the aforementioned primer combination, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain target nucleic acid-first primer-second primer hybrid 2;

任选地S3.将步骤S2获得的产物与前面所述的引物组合中的第三引物接触，所述第三引物的第六区域与所述第二引物的第二区域配对结合，获得靶标核酸-第一引物-第二引物-第三引物杂交体3。Optionally, S3. the product obtained in step S2 is contacted with the third primer in the primer combination described above, and the sixth region of the third primer is paired and bound with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3.

根据本发明一个具体的实施方案，所述核酸杂交方法进一步包括：步骤S1-S3中的接触是在含有碳酸乙烯酯的杂交反应液中进行的，所述杂交反应液中碳酸乙烯酯的浓度为5- 40％v/v，优选地，所述杂交反应液中碳酸乙烯酯的浓度为10％v/v。According to a specific embodiment of the present invention, the nucleic acid hybridization method further comprises: the contact in steps S1-S3 is carried out in a hybridization reaction solution containing ethylene carbonate, wherein the concentration of ethylene carbonate in the hybridization reaction solution is 5- 40% v/v, preferably, the concentration of ethylene carbonate in the hybridization reaction solution is 10% v/v.

根据本发明一个具体的实施方案，所述杂交反应液进一步包括硫酸葡聚糖和Triton-X-100，其中，所述硫酸葡聚糖的浓度为5-20％，所述Triton-X-100的浓度为0.05-0.5％。According to a specific embodiment of the present invention, the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

根据本发明一个具体的实施方案，所述杂交反应液为10％碳酸乙烯酯，10％硫酸葡聚糖，0.1％ Triton-X-100，溶剂：1X SSC。According to a specific embodiment of the present invention, the hybridization reaction solution is 10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, and the solvent is 1X SSC.

根据本发明一个具体的实施方案，所述核酸杂交方法进一步包括：在进行步骤S2之前，利用连接酶将步骤S1反应结束后体系中存在的第一引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合靶标核酸的第一引物。According to a specific embodiment of the present invention, the nucleic acid hybridization method further includes: before performing step S2, using a ligase to connect the first primer present in the system after the reaction of step S1 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess first primers that are not bound to the target nucleic acid.

根据本发明的实施方案，所述核酸杂交方法进一步包括：在进行步骤S3之前，利用连接酶将步骤S2反应结束后体系中存在的第二引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合第一引物的第二引物。According to an embodiment of the present invention, the nucleic acid hybridization method further includes: before performing step S3, using a ligase to connect the second primer present in the system after the reaction of step S2 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess second primers that are not bound to the first primer.

根据本发明一个具体的实施方案，所述核酸杂交方法进一步包括：在进行步骤S3之后，利用连接酶将步骤S3反应结束后体系中存在的第三引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合第二引物的第三引物。According to a specific embodiment of the present invention, the nucleic acid hybridization method further includes: after performing step S3, using a ligase to connect the third primer present in the system after the reaction of step S3 is completed to form a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess third primers that are not bound to the second primer.

需要说明的是，利用连接酶将引物连接成环，关于连接反应的温度和时间没有特别的限制，优选地在适合所述连接酶进行连接反应的温度下进行，可以在连接酶连接的最适温度上下浮动。连接时间只需要保证所有单链引物均连接成环即可。It should be noted that when ligating primers into a ring using a ligase, there are no particular restrictions on the temperature and time of the ligation reaction. The reaction is preferably carried out at a temperature suitable for the ligase, which may fluctuate above or below the optimal temperature for ligation. The ligation time only needs to ensure that all single-stranded primers are ligated into a ring.

根据本发明一个具体的实施方案，所述核酸杂交方法进一步包括：利用所述连接酶进行连接反应的反应温度为30-40℃，反应时间为5-30min。优选地，反应温度为37℃，反应时间为20min。According to a specific embodiment of the present invention, the nucleic acid hybridization method further comprises: using the ligase to perform a ligation reaction at a reaction temperature of 30-40° C. and a reaction time of 5-30 minutes. Preferably, the reaction temperature is 37° C. and the reaction time is 20 minutes.

根据本发明一个具体的实施方案，所述连接酶为T4连接酶。需要说明的是，本发明所述核酸杂交方法中用到的连接酶种类，并不限于T4连接酶，也可以是其他种类的连接酶，只要保证能够将引物的5’端和3’端首尾连接即可。According to a specific embodiment of the present invention, the ligase is T4 ligase. It should be noted that the type of ligase used in the nucleic acid hybridization method of the present invention is not limited to T4 ligase, and other types of ligases can also be used as long as they can ensure end-to-end ligation of the 5' and 3' ends of the primers.

根据本发明一个具体的实施方案，所述清洗液包括碳酸乙烯酯，所述碳酸乙烯酯在所述清洗液中的浓度为5-40％v/v。According to a specific embodiment of the present invention, the cleaning solution comprises ethylene carbonate, and the concentration of the ethylene carbonate in the cleaning solution is 5-40% v/v.

根据本发明一个具体的实施方案，所述清洗液进一步包括硫酸葡聚糖和Triton-X-100，其中，所述硫酸葡聚糖的浓度为5-20％，所述Triton-X-100的浓度为0.05-0.5％。According to a specific embodiment of the present invention, the cleaning solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

根据本发明一个具体的实施方案，步骤S1中配对结合的反应温度为30-40℃，反应时间为30-120min。According to a specific embodiment of the present invention, the reaction temperature for the pairing binding in step S1 is 30-40° C., and the reaction time is 30-120 min.

传统荧光原位杂交技术第一杂交引物需要过夜潮湿环境37℃孵育条件，而本发明的杂交方法仅需30-120min左右即可完成类似效果的高效反应。The first hybridization primer of the traditional fluorescence in situ hybridization technique needs to be incubated overnight in a humid environment at 37°C, while the hybridization method of the present invention only takes about 30-120 minutes to complete a high-efficiency reaction with similar effects.

根据本发明一个具体的实施方案，步骤S2中配对结合的反应温度为30-40℃，反应时间为20-40min。According to a specific embodiment of the present invention, the reaction temperature for the pairing binding in step S2 is 30-40° C., and the reaction time is 20-40 min.

根据本发明一个具体的实施方案，步骤S3中配对结合的反应温度为30-40℃，反应时间为20-40min。According to a specific embodiment of the present invention, the reaction temperature for the pairing binding in step S3 is 30-40° C., and the reaction time is 20-40 min.

根据本发明一个具体的实施方案，所述核酸杂交方法进一步包括：According to a specific embodiment of the present invention, the nucleic acid hybridization method further comprises:

Ⅰ.在步骤S3之后，继续重复步骤S2-S3至少N轮，N≥1，N为整数；或者，Ⅰ. After step S3, continue to repeat steps S2-S3 for at least N rounds, where N≥1 and N is an integer; or,

Ⅱ.在步骤S3之后，继续重复步骤S2-S3至少N’轮，N’≥0，N’为整数，且在最后一轮仅重复步骤S2。II. After step S3, continue to repeat steps S2-S3 for at least N' rounds, N'≥0, N' is an integer, and only repeat step S2 in the last round.

本发明提供一种检测靶标核酸的方法，所述方法包括：利用前面所述的核酸杂交方法，获得靶标核酸、第一引物、第二引物、任选的第三引物组成的杂交体，利用第一检测探针和/或第二检测探针检测所述杂交体，其中，所述第一检测探针包括与所述第二引物中的不与所述第一引物配对的区域的至少一部分配对结合的序列；所述第二检测探针包括与所述第三引物中的不与所述第二引物配对的区域的至少一部分配对结合的序列；所述第一检测探针和所述第二检测探针含有可检测信号的标记物。The present invention provides a method for detecting a target nucleic acid, comprising: using the aforementioned nucleic acid hybridization method to obtain a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer; and detecting the hybrid using a first detection probe and/or a second detection probe, wherein the first detection probe includes a sequence that pairs and binds to at least a portion of a region of the second primer that is not paired with the first primer; the second detection probe includes a sequence that pairs and binds to at least a portion of a region of the third primer that is not paired with the second primer; and the first detection probe and the second detection probe contain markers that can detect signals.

根据本发明一个具体的实施方案，所述第一检测探针包括与所述第二引物中的第二区域的至少一部分配对结合的序列；所述第一检测探针包括与所述第二引物中的第七片段的至少 一部分和第八片段的至少一部分配对结合的序列。According to a specific embodiment of the present invention, the first detection probe includes a sequence that pairs with at least a portion of the second region in the second primer; the first detection probe includes a sequence that pairs with at least a portion of the seventh fragment in the second primer. A sequence that pairs and binds with at least a portion of the eighth segment.

根据本发明一个具体的实施方案，所述第二检测探针包括与所述第三引物中的第三区域的至少一部分配对结合的序列；所述第二检测探针包括与所述第三引物中的第三片段的至少一部分和第四片段的至少一部分配对结合的序列。According to a specific embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer; the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

根据本发明一个具体的实施方案，所述方法进一步包括：According to a specific embodiment of the present invention, the method further comprises:

i.在步骤S2之后，以所述第一检测探针检测所述靶标核酸、第一引物以及第二引物组成的杂交体；或者i. After step S2, detecting the hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe; or

ⅱ.在步骤S3之后继续重复步骤S2-S3至少N轮的情况，则以所述第二检测探针检测所述靶标核酸、第一引物、第二引物以及第三引物组成的杂交体，任选地，利用所述第一检测探针进行辅助检测，其中N为自然数；或者ii. if steps S2-S3 are repeated for at least N rounds after step S3, the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer is detected with the second detection probe, and optionally, auxiliary detection is performed using the first detection probe, where N is a natural number; or

ⅱi.在步骤S3之后，继续重复步骤S2-S3至少N’轮，且在最后一轮仅重复步骤S2的情况，则以所述第一检测探针检测所述靶标核酸、第一引物、第二引物以及第三引物组成的杂交体，任选地，利用所述第二检测探针进行辅助检测，其中N’为正整数。ii. After step S3, continue to repeat steps S2-S3 for at least N' rounds, and in the last round, only repeat step S2, then use the first detection probe to detect the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer, and optionally, use the second detection probe for auxiliary detection, wherein N' is a positive integer.

根据本发明一个具体的实施方案，所述第一、二检测探针的探针长度分别独立地为13-23nt。According to a specific embodiment of the present invention, the lengths of the first and second detection probes are independently 13-23 nt.

本发明还提供一种核酸、蛋白质联合检测的方法，包括：The present invention also provides a method for combined detection of nucleic acids and proteins, comprising:

1)利用前面所述的检测靶标核酸的方法对含有核酸的待测生物样本进行核酸检测；以及1) performing nucleic acid detection on a biological sample containing nucleic acid using the aforementioned method for detecting target nucleic acid; and

2)对所述生物样本进行蛋白质荧光染色与成像。2) performing protein fluorescence staining and imaging on the biological sample.

根据本发明一个具体的实施方案，所述蛋白质荧光染色包括利用抗体进行蛋白质多轮染色。According to a specific embodiment of the present invention, the protein fluorescent staining comprises multiple rounds of protein staining using antibodies.

根据本发明的实施方案，当进行核酸、蛋白质联合检测时，含有核酸的待测生物样本优选地固定在固体支持物上。根据本发明一个具体的实施方案，所述待测生物样本为组织/细胞切片类样本。According to an embodiment of the present invention, when performing a combined nucleic acid and protein detection, the biological sample containing nucleic acid is preferably fixed on a solid support. According to a specific embodiment of the present invention, the biological sample is a tissue/cell section sample.

根据本发明一个具体的实施方案，本发明提供一种核酸检测引物组合，其包括：According to a specific embodiment of the present invention, the present invention provides a nucleic acid detection primer combination, which includes:

(1)第一引物，所述第一引物包括：a.两个与待测靶标核酸序列互补的配对区；和b.与待测靶标核酸序列不互补的非配对区，其中，a.中的两个配对区位于所述第一引物的两端，所述非配对区包括两组相同的序列，所述两组相同的序列分别与第一引物两端的配对区相连，且所述两组相同的序列之间通过第一间隔序列相连；(1) A first primer comprising: a. two pairing regions complementary to the target nucleic acid sequence to be detected; and b. a non-pairing region non-complementary to the target nucleic acid sequence to be detected, wherein the two pairing regions in a. are located at both ends of the first primer, and the non-pairing region comprises two groups of identical sequences, the two groups of identical sequences are respectively connected to the pairing regions at both ends of the first primer, and the two groups of identical sequences are connected by a first spacer sequence;

(2)第二引物，所述第二引物包括：c.与所述第一引物中的非配对区的两组相同的序列中的任一组互补配对的配对区；和d.与所述第一引物不互补的非配对区，其中，c.中的配对区包括两个序列且分别位于所述第二引物的两端，d.中的非配对区包括两组相同的序列，且分别与所述第二引物的两端的配对区相连，所述两组相同的序列之间通过第二间隔序列相连；(2) a second primer, comprising: c. a pairing region complementary to any one of the two identical sequences in the non-pairing region of the first primer; and d. a non-pairing region non-complementary to the first primer, wherein the pairing region in c. comprises two sequences and is located at both ends of the second primer, respectively; and the non-pairing region in d. comprises two identical sequences and is connected to the pairing regions at both ends of the second primer, respectively, and the two identical sequences are connected by a second spacer sequence;

(3)第三引物，所述第三引物包括：e.与所述第二引物中的非配对区的两组相同的序列中的任一组互补配对的配对区；和f.与所述第二引物不互补的非配对区，其中，e.中的配对区包括两个序列且分别位于所述第三引物的两端，f.中的非配对区包括两组相同的序列，且分别与所述第三引物的两端的配对区相连，所述两组相同的序列之间通过第三间隔序列相连，(3) a third primer, comprising: e. a pairing region complementary to any one of the two identical sequences in the non-pairing region of the second primer; and f. a non-pairing region non-complementary to the second primer, wherein the pairing region in e. comprises two sequences and is located at both ends of the third primer, respectively; the non-pairing region in f. comprises two identical sequences and is connected to the pairing regions at both ends of the third primer, respectively; and the two identical sequences are connected by a third spacer sequence.

其中，b.中非配对区的两组相同的序列与f.中非配对区的两组相同的序列相同。Among them, the two groups of identical sequences in the non-paired region in b. are identical to the two groups of identical sequences in the non-paired region in f.

根据本发明一个具体的实施方案，所述第一引物中，a.中的两个配对区的长度各自独立地为15-20nt。例如第一引物中，a.中的两个配对区的长度一个为15nt，另一个为16nt。“各自独立”即长度互不影响。两个配对区的长度可以相同也可以不同。针对不同的靶标基因的mRNA或DNA，根据该基因的序列长度可设置多个靶标位点，例如可设置10-30个位点，以实现在同一个靶标核酸上多个第一引物的结合，也即实现多个位点的信号放大。According to a specific embodiment of the present invention, in the first primer, the lengths of the two pairing regions in a. are independently 15-20 nt. For example, in the first primer, the lengths of the two pairing regions in a. are 15 nt and 16 nt respectively. "Independent of each other" means that the lengths do not affect each other. The lengths of the two pairing regions can be the same or different. For different target gene mRNA or DNA, multiple target sites can be set according to the sequence length of the gene, for example, 10-30 sites can be set to achieve the binding of multiple first primers on the same target nucleic acid, that is, to achieve signal amplification of multiple sites.

根据本发明一个具体的实施方案，本发明提供的引物组合在与靶标mRNA互补时，结构如图1所示，#1引物(第一引物)的第一片段和第二片段(二者组成第四(Ⅳ)区域)结合玻片上的mRNA，形成初级杂交信号；而#1引物除了含有第一片段(图中以数字“①”标出，其他片段以相同方式表示，仅数字不同)和第二片段之外，还包含两组分别相连的第三片段和第四片段(二者组成第一(Ⅰ)区域)，且其分别与两个#2引物对应第五和第六片段(二 者组成第五(Ⅴ)区域)互补结合；而每个#2引物不仅包含第五和第六片段，还包含2组分别相连的第七片段和第八片段(二者组成第二(Ⅱ)区域)，且其分别与两个#3引物对应第九片段和第十片段的互补结合；#3引物除了含有第九片段和第十片段之外，还包含两组与#1引物的第三片段和第四片段相同的序列，#3引物中含有的两组相连的第三片段和第四片段可用于与下一轮的第二引物的第五和第六片段互补结合。本发明通过多个#2引物与多个#3引物的互补结合，能够形成指数扩增状态。荧光信号的捕捉通过靶向结合有特定序列规律的#2或#3引物的15bp左右的读取序列(荧光探针)，能够更容易、更快速地检测目标核酸。对于每个引物序列，T4 DNA连接酶的作用是在每个扩增结束的阶段，连接此序列的头端和尾端成环。According to a specific embodiment of the present invention, when the primer combination provided by the present invention is complementary to the target mRNA, the structure is as shown in Figure 1, the first segment and the second segment of the #1 primer (the first primer) (the two segments constitute the fourth (IV) region) bind to the mRNA on the glass slide to form a primary hybridization signal; and in addition to containing the first segment (marked with the number "①" in the figure, the other segments are represented in the same way, only the numbers are different) and the second segment, the #1 primer also contains two groups of respectively connected third segments and fourth segments (the two segments constitute the first (I) region), and they respectively bind to the fifth and sixth segments (the second and third segments) corresponding to the two #2 primers. The fifth (V) region is formed by complementary binding; each #2 primer contains not only the fifth and sixth segments, but also two sets of connected seventh and eighth segments (the two together form the second (II) region), which complement each other with the corresponding ninth and tenth segments of the two #3 primers. In addition to the ninth and tenth segments, the #3 primer also contains two sets of sequences identical to the third and fourth segments of the #1 primer. The two sets of connected third and fourth segments contained in the #3 primer can be used to complementarily bind with the fifth and sixth segments of the second primer in the next round. The present invention can form an exponential amplification state through the complementary binding of multiple #2 primers and multiple #3 primers. The capture of fluorescent signals can be achieved by targeting the approximately 15bp reading sequence (fluorescent probe) of the #2 or #3 primer with specific sequence patterns, making it easier and faster to detect the target nucleic acid. For each primer sequence, the role of T4 DNA ligase is to connect the head and tail ends of this sequence to form a loop at the end of each amplification stage.

这样的引物序列设计，能保证每个层级的引物序列之间靶向结合，更易于捕捉；而双结合区域的设置方便扩增进一步放大，形成指数扩增的效果；同时使用T4 DNA连接酶而不是点击化学的方法来成环，使成本大大降低。This type of primer sequence design can ensure targeted binding between primer sequences at each level, making capture easier; the setting of the double binding region facilitates further amplification, forming an exponential amplification effect; at the same time, the use of T4 DNA ligase instead of click chemistry to form a ring greatly reduces costs.

需要说明的是，根据本发明一个具体的实施方案，如在图1中，第一片段与相邻的第三片段之间、第二片段与相邻的第四片段之间、第五片段与相邻的第七片段之间、第六片段与相邻的第八片段之间、第九片段与相邻的第三片段之间、第十片段与相邻的第四片段之间可以各自独立地具有间隔序列或者直接相连，当具有间隔序列时，间隔序列长度优选为2-5nt。It should be noted that, according to a specific embodiment of the present invention, as shown in Figure 1, the first fragment and the adjacent third fragment, the second fragment and the adjacent fourth fragment, the fifth fragment and the adjacent seventh fragment, the sixth fragment and the adjacent eighth fragment, the ninth fragment and the adjacent third fragment, and the tenth fragment and the adjacent fourth fragment can each independently have a spacer sequence or be directly connected. When there is a spacer sequence, the length of the spacer sequence is preferably 2-5nt.

根据本发明一个具体的实施方案，所述待测靶标核酸为单链DNA或RNA。RNA可以是组织切片中细胞内直接转录的mRNA，而单链DNA可以是源自一些肿瘤细胞的单链DNA。According to a specific embodiment of the present invention, the target nucleic acid to be detected is single-stranded DNA or RNA. The RNA can be mRNA directly transcribed in cells in a tissue section, and the single-stranded DNA can be single-stranded DNA derived from some tumor cells.

根据本发明一个具体的实施方案，本发明提供的核酸引物组合、套装可用于以下用途：According to a specific embodiment of the present invention, the nucleic acid primer combination and kit provided by the present invention can be used for the following purposes:

a.核酸原位杂交；a. Nucleic acid in situ hybridization;

b.核酸、蛋白质联合检测；b. Combined detection of nucleic acid and protein;

c.组织图谱构建；c. Organizational map construction;

d.确定组织中细胞分布；d. Determine the distribution of cells in tissues;

e.数字病理成像和分析；e. Digital pathology imaging and analysis;

f.生物标志物的筛选和发现。f. Biomarker screening and discovery.

根据本发明一个具体的实施方案，进行蛋白质和核酸(如RNA)联合检测优选在组织中进行，通过定位表达特定标记物的细胞位置，可以构建组织图谱，确定组织中细胞分布，以及筛选生物标志物等。本发明的核酸引物组合、套装也可用于数字病例成像。According to a specific embodiment of the present invention, combined detection of proteins and nucleic acids (such as RNA) is preferably performed in tissues. By locating cells expressing specific markers, tissue maps can be constructed, cell distribution within tissues can be determined, and biomarkers can be screened. The nucleic acid primer combinations and kits of the present invention can also be used for digital medical imaging.

根据本发明一个具体的实施方案，本发明提供一种目标核酸原位杂交方法，所述方法包括利用前面所述的核酸检测引物组合与目标核酸互补配对并放大目标核酸信号，利用与所述第二引物或第三引物的非配对区互补的荧光标记探针检测所述目标核酸。其中，所述目标核酸为单链DNA或RNA；所述荧光标记探针的探针长度为13-23nt。According to a specific embodiment of the present invention, a method for in situ hybridization of a target nucleic acid is provided, comprising using the aforementioned nucleic acid detection primer combination to complementarily pair with a target nucleic acid and amplify the target nucleic acid signal, and detecting the target nucleic acid using a fluorescently labeled probe complementary to an unpaired region of the second or third primer. The target nucleic acid is single-stranded DNA or RNA, and the fluorescently labeled probe has a probe length of 13-23 nt.

根据本发明一个具体的实施方案，本发明提供一种荧光原位杂交方法，其包括以下步骤：According to a specific embodiment of the present invention, the present invention provides a fluorescence in situ hybridization method, which comprises the following steps:

1)制备待测组织切片；1) Preparing tissue sections to be tested;

2)将前面所述的核酸检测引物组合中的第一引物与所述待测组织切片中组织接触，进行所述第一引物与待测组织中靶标RNA的互补配对反应；2) contacting the first primer in the aforementioned nucleic acid detection primer combination with the tissue in the tissue section to be tested, and performing a complementary pairing reaction between the first primer and the target RNA in the tissue to be tested;

3)利用连接酶将2)中第一引物连接成环，去除多余的未结合靶标RNA的第一引物，以便获得含有靶标RNA-已成环第一引物的待测组织切片；3) using a ligase to link the first primers in 2) into a ring, removing excess first primers that have not bound to the target RNA, thereby obtaining a tissue section to be tested containing the target RNA-circularized first primer;

4)将前面所述的核酸检测引物组合中的第二引物与经步骤3)处理的待测组织接触，以便进行第二引物与经步骤3)处理的待测组织中已成环第一引物的互补配对反应；4) contacting the second primer in the aforementioned nucleic acid detection primer combination with the tissue to be tested treated in step 3) to allow for a complementary pairing reaction between the second primer and the first primer that has been circularized in the tissue to be tested treated in step 3);

5)利用连接酶将4)中第二引物连接成环，去除多余的未结合第一引物的第二引物，以便获得含有靶标RNA-已成环第一引物-已成环第二引物的待测组织切片；5) ligating the second primer in 4) into a ring using a ligase, and removing excess second primer not bound to the first primer, thereby obtaining a tissue section to be tested containing target RNA-circularized first primer-circularized second primer;

6)将前面所述的核酸检测引物组合中的第三引物与经步骤5)处理的待测组织接触，以便进行第三引物与经步骤5)处理的待测组织中已成环第二引物的互补配对反应；6) contacting the third primer in the aforementioned nucleic acid detection primer combination with the tissue to be tested treated in step 5) to allow for a complementary pairing reaction between the third primer and the looped second primer in the tissue to be tested treated in step 5);

7)利用连接酶将6)中第三引物连接成环，去除多余的未结合第二引物的第三引物，以便获得含有靶标RNA-已成环第一引物-已成环第二引物-已成环第三引物的待测组织切片；以及 7) using a ligase to link the third primer in step 6) into a ring, removing excess third primer not bound to the second primer, thereby obtaining a tissue section to be tested containing target RNA-circularized first primer-circularized second primer-circularized third primer; and

8)利用荧光标记探针与经7)处理的待测组织接触，以便进行荧光标记探针与经步骤7)处理的待测组织中已成环第三引物的互补配对反应，去除多余未结合的荧光标记探针，并进行待测组织切片成像拍照。8) contacting the tissue to be tested treated in step 7) with the fluorescently labeled probe to allow for a complementary pairing reaction between the fluorescently labeled probe and the third primer that has been circularized in the tissue to be tested treated in step 7), removing excess unbound fluorescently labeled probe, and imaging the tissue section to be tested.

根据本发明一个具体的实施方案，步骤2)、4)、6)中引物与待测组织是在含有碳酸乙烯酯的杂交反应液中接触的。所述杂交反应液中碳酸乙烯酯的浓度为5-40％v/v。碳酸乙烯酯加入缓冲液中，进一步缩短了#1与靶标mRNA孵育时间。According to a specific embodiment of the present invention, in steps 2), 4), and 6), the primers and the tissue to be tested are contacted in a hybridization reaction solution containing ethylene carbonate. The concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v/v. The addition of ethylene carbonate to the buffer further shortens the incubation time between #1 and the target mRNA.

根据本发明一个具体的实施方案，本发明提供一种对组织切片进行RNA、蛋白质联合检测的方法，其包括以下步骤：According to a specific embodiment of the present invention, the present invention provides a method for joint detection of RNA and protein in tissue sections, comprising the following steps:

(Ⅰ)利用前面所述的荧光原位杂交方法对所述组织切片进行RNA荧光原位杂交，对经处理后的组织切片进行拍照记录；以及(I) performing RNA fluorescence in situ hybridization on the tissue sections using the aforementioned fluorescence in situ hybridization method, and photographing and recording the processed tissue sections; and

(Ⅱ)对拍照记录后的组织切片直接进行组织蛋白质荧光染色与成像。(II) Directly perform tissue protein fluorescence staining and imaging on the tissue sections after photographing.

根据本发明一个具体的实施方案，所述组织蛋白质荧光染色包括利用抗体进行蛋白质多轮染色。According to a specific embodiment of the present invention, the tissue protein fluorescent staining includes multiple rounds of protein staining using antibodies.

本发明提供的核酸原位杂交方法，高效、省时、廉价且能够实现RNA、蛋白联检。RNA荧光检测可进行信号放大，无需过多使用荧光探针，缩短了孵育时间。本发明提供的核酸原位杂交方法，使用的引物组合中仅需要改变第一引物的序列，而第二、第三引物为通用引物，且对不同RNA目标可使用同一种荧光探针检测，大大降低成本。在RNA检测后，可直接进行蛋白染色，重复定位实现联检。The nucleic acid in situ hybridization method provided by the present invention is efficient, time-saving, inexpensive, and capable of combined RNA and protein detection. RNA fluorescence detection can amplify the signal without the need for excessive use of fluorescent probes, thus shortening the incubation time. The nucleic acid in situ hybridization method provided by the present invention uses a primer combination that only requires changing the sequence of the first primer, while the second and third primers are universal primers, and the same fluorescent probe can be used for detection of different RNA targets, greatly reducing costs. After RNA detection, protein staining can be performed directly, and repeated positioning can be used to achieve combined detection.

本领域技术人员将会理解，下面的实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。Those skilled in the art will understand that the following examples are merely illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications were used. Where the manufacturer of the reagents or instruments used is not specified, they are all commercially available conventional products.

实施例1：RNA原位杂交Example 1: RNA in situ hybridization

对小鼠肿瘤组织进行RNA原位杂交检测，此肿瘤组织为MDA-MB231肿瘤模型，蜡块购自武汉赛维尔。RNA in situ hybridization was performed on mouse tumor tissue, which was an MDA-MB231 tumor model. The paraffin blocks were purchased from Wuhan Saiweier.

1.石蜡切片的预处理：1. Pretreatment of paraffin sections:

a.肿瘤组织蜡块用石蜡切片机切片后放入40-45℃水浴锅；a. Slice the tumor tissue paraffin block using a paraffin microtome and place it in a 40-45°C water bath.

b.使用表面处理(Poly-l-lysine，APTS，Gelatin，Gelatin Chrome Alum等)后的载玻片进行捞片并竖直静置过夜；b. Use a surface-treated slide (Poly-l-lysine, APTS, Gelatin, Gelatin Chrome Alum, etc.) to fish out the slide and place it upright overnight.

c.使用二甲苯(Xylene)进行脱蜡处理，时间为3小时至过夜；c. Dewaxing with xylene for 3 hours to overnight;

d.按顺序放入无水乙醇、95％乙醇、70％乙醇、50％乙醇、超纯水(每种溶液10-30分钟)进行水化处理；d. Add anhydrous ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and ultrapure water in sequence (10-30 minutes for each solution) for hydration;

e.切片放入抗原修复液中进行高温(95℃)进行10-20分钟抗原修复。e. Place the sections in antigen retrieval solution and perform antigen retrieval at high temperature (95°C) for 10-20 minutes.

2.RNA荧光原位杂交检测：2. RNA fluorescence in situ hybridization detection:

a.#1、#2、#3反应引物(探针引物均使用TE稀释为100μM)与读取引物溶解与稀释：分别加不同量的TE缓冲液溶解引物，保证引物完全溶解，之后按照1：100浓度(或所需浓度，按计算来确定)取EP管稀释上述各种引物，并标记为稀释引物管；a. Dissolve and dilute the reaction primers #1, #2, and #3 (probe primers are all diluted to 100 μM using TE) and the read primer: Dissolve each primer in varying amounts of TE buffer until completely dissolved. Then, dilute each primer in an EP tube at a 1:100 concentration (or the desired concentration, determined by calculation) and label it as the diluted primer tube.

b.常规清洗缓冲液配制，读取冲洗缓冲液配制：需先稀释20X SSC至1X SSC备用，溶剂：超纯水；常规清洗缓冲液配制(10％碳酸乙烯酯，溶剂使用1X SSC)；读取冲洗缓冲液＝1XSSC+0.1％ Triton-X-100；b. Prepare the conventional wash buffer and read rinse buffer: dilute 20X SSC to 1X SSC using ultrapure water. Prepare the conventional wash buffer (10% ethylene carbonate, 1X SSC as solvent). Read rinse buffer = 1X SSC + 0.1% Triton-X-100.

c.杂交反应溶液配制,读取缓冲液配制：10％杂交反应液(10％碳酸乙烯酯，10％硫酸葡聚糖，0.1％ Triton-X-100，溶剂：1X SSC)，用于#1引物-组织杂交以及用于#2、#3引物扩增；10％杂交反应液(10％碳酸乙烯酯，10％硫酸葡聚糖，0.1％ Triton-X-100，溶剂：1X SSC)，用于洗脱非特异性结合的引物结合；读取缓冲液：10％硫酸葡聚糖+0.1％ Tx-100+5％碳酸乙烯酯；T4 DNA连接酶反应液：1ul T4 DNA连接酶+99ul 1X酶缓冲液；c. Preparation of hybridization reaction solution and read buffer: 10% hybridization reaction solution (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC) for #1 primer-tissue hybridization and #2 and #3 primer amplification; 10% hybridization reaction solution (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC) for eluting non-specifically bound primers; read buffer: 10% dextran sulfate + 0.1% Tx-100 + 5% ethylene carbonate; T4 DNA ligase reaction solution: 1ul T4 DNA ligase + 99ul 1X enzyme buffer;

d.计算#1、#2、#3、读取、T4连接酶总反应体系制备各原料量并配制，其配比如c中所示，各反应液体系约200μL；d. Calculate #1, #2, #3, read, and prepare the total amount of each raw material in the T4 ligase reaction system. The formula is as shown in c, and each reaction solution is about 200 μL.

e.正式反应：热盘打开并调制37℃加热状态，放置已有折痕的封口膜于热盘中央，封口 膜中间加入1ml常规清洗缓冲液，倒扣放置组织切片于封口膜中央，开始反应计时；e. Formal reaction: Open the hot plate and adjust the temperature to 37℃. Place the creased sealing film in the center of the hot plate and seal it. Add 1 ml of regular washing buffer to the middle of the membrane, place the tissue section upside down in the center of the sealing membrane, and start the reaction timer;

f.结束计时后，弃去清洗液，并小心转移倒扣玻片至另一滴有100μL#1总反应体系(10％碳酸乙烯酯,10％硫酸葡聚糖,溶剂：1X SSC，以及#1探针各3.6nM)的封口膜中央，注意转移玻片时不要刮到组织，继续37℃反应90min或150min；f. After the timer expires, discard the wash solution and carefully transfer the inverted slide to the center of another drop of sealing film containing 100 μL of the total reaction system #1 (10% ethylene carbonate, 10% dextran sulfate, solvent: 1X SSC, and 3.6 nM each of probe #1). Be careful not to scrape the tissue when transferring the slide. Continue the reaction at 37°C for 90 min or 150 min.

g.T4 DNA连接酶反应液添加至封口膜中央，连接成环，继续37℃反应20min；g. Add T4 DNA ligase reaction solution to the center of the sealing membrane to form a ring. Continue the reaction at 37°C for 20 minutes.

h.3ml常规清洗缓冲液清洗玻片，同样加至同一封口膜，37℃清洗10min；h. Wash the slides with 3 ml of conventional wash buffer, add to the same sealing film, and wash at 37°C for 10 minutes;

i.转移倒扣玻片至另一滴有#2总反应体系(10％碳酸乙烯酯,10％硫酸葡聚糖,0.1％Triton-X-100,溶剂：1X SSC，以及#2探针20nM)的封口膜中央，热盘37℃反应扩增30min；i. Transfer the inverted slide to the center of another drop of sealing film containing the total reaction system #2 (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC, and 20nM probe #2) and amplify on a hot plate at 37°C for 30 minutes.

j.T4 DNA连接酶反应液添加至封口膜中央，连接成环，继续37℃反应20min；j. Add T4 DNA ligase reaction solution to the center of the sealing membrane to form a ring. Continue the reaction at 37°C for 20 minutes.

k.3ml常规清洗液清洗玻片，同样加至同一封口膜，37℃清洗10min；k. Wash the slides with 3 ml of conventional cleaning solution, add it to the same sealing film, and wash at 37°C for 10 minutes;

l.转移倒扣玻片至另一滴有#3总反应体系(10％碳酸乙烯酯,10％硫酸葡聚糖,0.1％Triton-X-100,溶剂：1X SSC，以及#3探针20nM)的封口膜中央，热盘37℃反应扩增30min；Transfer the inverted slide to the center of another drop of sealing film containing the total reaction system #3 (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC, and 20 nM probe #3) and amplify on a hot plate at 37°C for 30 min.

m.T4 DNA连接酶反应液添加至封口膜中央，连接成环，继续37℃反应至少20min；Add T4 DNA ligase reaction solution to the center of the sealing membrane to form a ring. Continue the reaction at 37°C for at least 20 minutes.

n.3ml常规清洗液清洗玻片,同样加至同一封口膜，37℃清洗10min；如需多轮扩增#2、#3，则需重复i.-n.步骤几轮，直到可以继续进行读取荧光标记为止；Wash the slides with 3 ml of conventional cleaning solution, then apply to the same sealing film and wash at 37°C for 10 minutes. If multiple rounds of amplification #2 and #3 are required, repeat steps i.-n. several times until the fluorescent markers can be read.

o.读取缓冲液体系计算、配制完成后，转移倒扣玻片至另一滴有读取缓冲液反应体系的封口膜中央，常温孵育20min；根据扩增轮次数量调整读取缓冲液的反应体系浓度；After calculating and preparing the read buffer system, transfer the inverted slide to the center of another drop of sealing film containing the read buffer reaction system and incubate at room temperature for 20 minutes. Adjust the concentration of the read buffer reaction system according to the number of amplification rounds;

p.读取冲洗缓冲液常温清洗玻片1min 2次，保存玻片至4℃1XSSC内或载片加入成像缓冲液上机拍照染色情况。p. Read the rinsing buffer and wash the slides twice at room temperature for 1 minute. Store the slides in 1XSSC at 4°C or add imaging buffer to the slides and take photos of the staining status.

进行RNA荧光原位杂交所用到的引物如下表1所示，以CD8 mRNA作为检测靶标设计多个引物#1-1～#1-16组合，采用#2、#3进行信号放大，利用#2读取探针、#3读取探针进行信号检测：The primers used for RNA fluorescence in situ hybridization are shown in Table 1 below. Multiple primer combinations #1-1 to #1-16 were designed using CD8 mRNA as the detection target. Primers #2 and #3 were used for signal amplification, and read probes #2 and #3 were used for signal detection:

表1引物及探针序列

Table 1 Primer and probe sequences

RNA荧光原位杂交检测结果如图2所示，表明利用本发明的#1-#3引物组合，能够大大缩短荧光读取和检测RNA杂交信号的孵育时间，能够快速地检测靶标RNA的原位表达。The results of RNA fluorescence in situ hybridization detection are shown in FIG2 , indicating that the use of the primer combination #1-#3 of the present invention can greatly shorten the incubation time for fluorescence reading and detecting RNA hybridization signals, and can quickly detect the in situ expression of target RNA.

发明人对#1引物反应时间以及读取缓冲液中探针浓度进行了对比实验，如图2所示，分成了三个实验组，分别为一级探针90min孵育时间+10nM检测探针；一级探针90min孵育时间+20nM检测探针；一级探针150min孵育时间+20nM检测探针，这三组实验方案的其他所有条件均相同；结果表明，最佳读取探针浓度为20nM，#1反应体系孵育时间为90分钟。The inventors conducted a comparative experiment on the reaction time of primer #1 and the probe concentration in the reading buffer. As shown in Figure 2, the three experimental groups were divided into: 90-minute incubation time of the primary probe + 10 nM detection probe; 90-minute incubation time of the primary probe + 20 nM detection probe; and 150-minute incubation time of the primary probe + 20 nM detection probe. All other conditions of the three experimental schemes were the same. The results showed that the optimal reading probe concentration was 20 nM and the incubation time of the #1 reaction system was 90 minutes.

进一步地，发明人对“2.RNA荧光原位杂交检测中的步骤c”中的杂交反应液成分进行了对比实验，其中一组为含有10％碳酸乙烯酯的反应溶液，另一组为将10％碳酸乙烯酯替换为10％聚乙烯胺的反应溶液；读取探针浓度为20nM，#1反应体系孵育时间为90分钟。结果如图3所 示，相同条件下使用10％聚乙烯胺进行的杂交实验无法提供对应的点状FISH信号，仅显示自发荧光的非特异性信号，表明聚乙烯胺染色效果要弱于碳酸乙烯酯。Furthermore, the inventors conducted a comparative experiment on the hybridization reaction solution components in "2. Step c of RNA fluorescence in situ hybridization detection". One group contained a reaction solution containing 10% ethylene carbonate, and the other group replaced 10% ethylene carbonate with 10% polyvinylamine. The read probe concentration was 20nM, and the incubation time of reaction system #1 was 90 minutes. The results are shown in Figure 3. The results show that the hybridization experiment using 10% polyvinylamine under the same conditions could not provide corresponding punctate FISH signals, but only showed nonspecific signals of autofluorescence, indicating that the staining effect of polyvinylamine was weaker than that of ethylene carbonate.

实施例2 RNA原位杂交和蛋白质荧光染色Example 2 RNA in situ hybridization and protein fluorescence staining

对小鼠肿瘤组织进行RNA原位杂交和组织蛋白质荧光染色，此肿瘤组织为MDA-MB231肿瘤模型，蜡块购自武汉赛维尔。RNA in situ hybridization and tissue protein fluorescence staining were performed on mouse tumor tissue. The tumor tissue was an MDA-MB231 tumor model, and the paraffin blocks were purchased from Wuhan Saiweier.

1.石蜡切片的预处理：操作步骤与实施例1的“1.石蜡切片的预处理”相同。1. Pretreatment of paraffin sections: The operation steps are the same as those in “1. Pretreatment of paraffin sections” in Example 1.

2.RNA荧光原位杂交检测：操作步骤与实施例1的“2.RNA荧光原位杂交检测”相同，其中，在步骤f.中反应时间为90min。2. RNA fluorescence in situ hybridization detection: The operation steps are the same as those in "2. RNA fluorescence in situ hybridization detection" of Example 1, wherein the reaction time in step f is 90 minutes.

进行RNA荧光原位杂交所用到的引物如实施例1中表1所示，以CD8 mRNA作为检测靶标设计多个引物#1-1～#1-16组合，采用#2、#3进行信号放大，利用#2读取探针、#3读取探针进行信号检测。The primers used for RNA fluorescence in situ hybridization are shown in Table 1 of Example 1. Multiple primer combinations #1-1 to #1-16 were designed using CD8 mRNA as the detection target. #2 and #3 were used for signal amplification, and #2 and #3 reading probes were used for signal detection.

RNA荧光原位杂交检测结果如图2所示，表明利用本发明的#1-#3引物组合，能够大大缩短荧光读取和检测RNA杂交信号的孵育时间，能够快速地检测靶标RNA的原位表达。The results of RNA fluorescence in situ hybridization detection are shown in FIG2 , indicating that the use of the primer combination #1-#3 of the present invention can greatly shorten the incubation time for fluorescence reading and detecting RNA hybridization signals, and can quickly detect the in situ expression of target RNA.

3.对原位杂交后的玻片拍照后直接进行蛋白全自动化染拍，具体地，包括制作流道，并对进行原位杂交后的玻片继续进行免疫荧光蛋白染色，步骤如下：3. After taking a photo of the slide after in situ hybridization, perform fully automated protein staining directly. Specifically, this includes making a flow channel and continuing to perform immunofluorescence protein staining on the slide after in situ hybridization. The steps are as follows:

a.加入0.5％ TritonX-100进行15分钟打孔；a. Add 0.5% Triton X-100 and perform perforation for 15 minutes;

b.使用清洗液进行5次清洗(时间：60秒，流速：1200微升每分钟)；b. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl per minute);

c.加入成像缓冲液(0.2mg/ml trolox)并捕获原始背景图片；c. Add imaging buffer (0.2 mg/ml Trolox) and capture the original background image;

d.使用清洗液(PBS)进行5次清洗(时间：60秒，流速：1200微升每分钟)；d. Wash five times with PBS (time: 60 seconds, flow rate: 1200 μl/min);

e.加入抗体洗脱液(0.8％Beta-ME、62.5mM Tris-HCl、1％SDS)(56℃，10分钟)；e. Add antibody elution buffer (0.8% Beta-ME, 62.5 mM Tris-HCl, 1% SDS) (56°C, 10 minutes);

f.使用清洗液进行5次清洗(时间：60秒，流速：1200微升每分钟)；f. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl per minute);

g.加入义翘(#10084-T24)PD-L1一抗(37℃，10分钟)；g. Add Sino-Biological (#10084-T24) PD-L1 primary antibody (37°C, 10 minutes);

h.使用清洗液进行5次清洗(时间：60秒，流速：1200微升每分钟)；h. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl per minute);

i.加入赛默飞(#A11009)AF532羊抗兔二抗(37℃，10分钟)；i. Add Thermo Fisher Scientific (#A11009) AF532 goat anti-rabbit secondary antibody (37°C, 10 minutes);

j.使用清洗液进行5次清洗(时间：60秒，流速：1200微升每分钟)j. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl/min)

k.重复步骤3-10，进行多轮抗体的免疫荧光染色。k. Repeat steps 3-10 for multiple rounds of immunofluorescence staining with antibodies.

免疫荧光蛋白染色结果如图4所示，表明在完成了mRNA杂交实验后，可使用抗体对组织上蛋白进一步进行免疫荧光染色，说明采用本发明特定的引物组合，并利用经优化后的反应体系，能够成功的进行核酸、蛋白质联合检测。The results of immunofluorescence protein staining are shown in FIG4 , indicating that after completing the mRNA hybridization experiment, antibodies can be used to further perform immunofluorescence staining on proteins on the tissue, indicating that the use of the specific primer combination of the present invention and the optimized reaction system can successfully perform nucleic acid and protein joint detection.

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、“一些实施方案”或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。 Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims (26)

一种引物组合，其包括：A primer combination comprising: (1)一种或多种第一引物，所述第一引物包括：与靶标核酸的片段配对结合的第四区域，以及至少一个与所述靶标核酸不配对结合的第一区域；(1) one or more first primers, each of which comprises: a fourth region that binds to a target nucleic acid fragment and at least one first region that does not bind to the target nucleic acid; (2)第二引物，所述第二引物包括：与所述第一区域配对结合的第五区域，以及至少两个不与所述靶标核酸配对结合的第二区域；(2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid; (3)第三引物，所述第三引物包括：与所述第二区域配对结合的第六区域，以及至少两个不与所述靶标核酸配对结合的第三区域。(3) A third primer, comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid. 根据权利要求1所述的引物组合，其中，所述第一引物包括：与靶标核酸的片段配对结合的第四区域，以及至少两个与所述靶标核酸的序列不配对结合的第一区域。The primer combination according to claim 1, wherein the first primer comprises: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that do not pair and bind to a sequence of the target nucleic acid. 根据权利要求1或2所述的引物组合，其中，所述第二区域不与所述第一区域配对结合；所述第三区域不与所述第二区域配对结合。The primer combination according to claim 1 or 2, wherein the second region does not pair with the first region; and the third region does not pair with the second region. 根据权利要求1-3中任一项所述的引物组合，其中，The primer combination according to any one of claims 1 to 3, wherein 所述第四区域包括第一片段和第二片段，所述第一片段和第二片段分别位于所述第一引物的两端，所述第一区域位于所述第一片段和第二片段之间；The fourth region includes a first fragment and a second fragment, the first fragment and the second fragment are respectively located at two ends of the first primer, and the first region is located between the first fragment and the second fragment; 每一个所述第一区域至少包括第三片段和第四片段；Each of the first regions includes at least a third segment and a fourth segment; 所述第五区域包括第五片段和第六片段，所述第五片段和第六片段分别位于所述第二引物的两端，所述第二区域位于所述第五片段和第六片段之间；The fifth region includes a fifth segment and a sixth segment, the fifth segment and the sixth segment are respectively located at two ends of the second primer, and the second region is located between the fifth segment and the sixth segment; 每一个所述第二区域至少包括第七片段和第八片段；Each of the second regions includes at least a seventh segment and an eighth segment; 所述第六区域包括第九片段和第十片段，所述第九片段和第十片段分别位于所述第三引物的两端，所述第三区域位于所述第九片段和第十片段之间；The sixth region includes a ninth segment and a tenth segment, the ninth segment and the tenth segment are located at both ends of the third primer, respectively, and the third region is located between the ninth segment and the tenth segment; 每一个所述第三区域至少包括所述第三片段和第四片段。Each of the third regions includes at least the third segment and the fourth segment. 根据权利要求1-4中任一项所述的引物组合，其包括多种第一引物，其中每一种所述第一引物的第四区域与不同靶标核酸的片段或相同靶标核酸的不同片段配对结合。The primer combination according to any one of claims 1 to 4, comprising a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid. 根据权利要求1-5中任一项所述的引物组合，其中，所述第一引物的5’端的第一个碱基与3’端的最后一个碱基对应与其配对结合的所述靶标核酸的片段上相邻的碱基。The primer combination according to any one of claims 1 to 5, wherein the first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the fragment of the target nucleic acid with which they are paired and bound. 根据权利要求4-6中任一项所述的引物组合，其中，所述第一片段和第二片段的长度各自独立地为15-20nt；The primer combination according to any one of claims 4 to 6, wherein the length of the first fragment and the second fragment are each independently 15-20 nt; 任选地，所述第三片段、第四片段、第五片段、第六片段、第七片段、第八片段、第九片段和第十片段的长度各自独立地为8-20nt，优选10-16nt。Optionally, the lengths of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment are each independently 8-20 nt, preferably 10-16 nt. 根据权利要求1-7中任一项所述的引物组合，其中，所述第一引物包括一个以上的所述第一区域，各个第一区域之间通过第一间隔序列相连；The primer combination according to any one of claims 1 to 7, wherein the first primer comprises more than one first region, and the first regions are connected by a first spacer sequence; 任选地，在第二引物中，各个所述第二区域之间通过第二间隔序列相连；Optionally, in the second primer, each of the second regions is connected by a second spacer sequence; 任选地，在第三引物中，各个所述第三区域之间通过第三间隔序列相连；Optionally, in the third primer, each of the third regions is connected by a third spacer sequence; 任选地，所述第一间隔序列、所述第二间隔序列和所述第三间隔序列的序列长度至少为1nt，优选1-30nt，进一步优选2-5nt。Optionally, the sequence length of the first spacer sequence, the second spacer sequence and the third spacer sequence is at least 1 nt, preferably 1-30 nt, and further preferably 2-5 nt. 一种套装，其包括：权利要求1-8中任一项所述的引物组合，以及第一检测探针和/或第二检测探针，A kit comprising: the primer combination according to any one of claims 1 to 8, and a first detection probe and/or a second detection probe, 其中，所述第一检测探针包括与所述第二引物中的不与所述第一引物配对的区域的至少一部分配对结合的序列；wherein the first detection probe comprises a sequence that pairs and binds to at least a portion of the region of the second primer that does not pair with the first primer; 所述第二检测探针包括与所述第三引物中的不与所述第二引物配对的区域的至少一部分配对结合的序列；The second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that does not pair with the second primer; 所述第一检测探针和所述第二检测探针含有可检测信号的标记物。The first detection probe and the second detection probe contain a label capable of generating a detectable signal. 根据权利要求9所述的套装，其中，所述第一检测探针包括与所述第二引物中的第二区域的至少一部分配对结合的序列；The kit according to claim 9, wherein the first detection probe comprises a sequence that pairs and binds to at least a portion of the second region in the second primer; 任选地，所述第一检测探针包括与所述第二引物中的第七片段的至少一部分和第八片段 的至少一部分配对结合的序列。Optionally, the first detection probe comprises at least a portion of the seventh segment and the eighth segment of the second primer. at least a portion of the paired binding sequence. 根据权利要求9或10所述的套装，其中，所述第二检测探针包括与所述第三引物中的第三区域的至少一部分配对结合的序列；The kit according to claim 9 or 10, wherein the second detection probe comprises a sequence that pairs and binds to at least a portion of the third region in the third primer; 任选地，所述第二检测探针包括与所述第三引物中的第三片段的至少一部分和第四片段的至少一部分配对结合的序列。Optionally, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer. 根据权利要求9-11中任一项所述的套装，其中，所述标记物包括放射性标记物或非放射性标记物；The kit according to any one of claims 9 to 11, wherein the label comprises a radioactive label or a non-radioactive label; 任选地，所述非放射性标记物包括荧光基团。Optionally, the non-radioactive label comprises a fluorescent group. 一种核酸杂交方法，其包括：A nucleic acid hybridization method comprising: S1.将含有靶标核酸的生物样本与权利要求1-8中任一项所述的引物组合中的一种或多种第一引物接触，所述一种或多种第一引物中的第四区域与靶标核酸的片段配对结合，获得靶标核酸-第一引物杂交体1；S1. contacting a biological sample containing a target nucleic acid with one or more first primers of the primer combination of any one of claims 1 to 8, wherein the fourth region of the one or more first primers pairs and binds to a fragment of the target nucleic acid to obtain a target nucleic acid-first primer hybrid 1; S2.将步骤S1获得的产物与权利要求1-8中任一项所述的引物组合中的第二引物接触，所述第二引物的第五区域与所述第一引物的第一区域配对结合，获得靶标核酸-第一引物-第二引物杂交体2；S2. contacting the product obtained in step S1 with the second primer in the primer combination of any one of claims 1 to 8, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain target nucleic acid-first primer-second primer hybrid 2; 任选地S3.将步骤S2获得的产物与权利要求1-8中任一项所述的引物组合中的第三引物接触，所述第三引物的第六区域与所述第二引物的第二区域配对结合，获得靶标核酸-第一引物-第二引物-第三引物杂交体3。Optionally, S3. contacting the product obtained in step S2 with the third primer in the primer combination of any one of claims 1 to 8, and pairing and binding the sixth region of the third primer with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3. 根据权利要求13所述的核酸杂交方法，其中，所述方法进一步包括：步骤S1-S3中的接触是在含有碳酸乙烯酯的杂交反应液中进行的，所述杂交反应液中碳酸乙烯酯的浓度为5-40％v/v；The nucleic acid hybridization method according to claim 13, wherein the method further comprises: the contacting in steps S1-S3 is performed in a hybridization reaction solution containing ethylene carbonate, wherein the concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v/v; 任选地，所述杂交反应液进一步包括硫酸葡聚糖和Triton-X-100，其中，所述硫酸葡聚糖的浓度为5-20％，所述Triton-X-100的浓度为0.05-0.5％。Optionally, the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%. 根据权利要求13或14所述的核酸杂交方法，其中，所述方法进一步包括：在进行步骤S2之前，利用连接酶将步骤S1反应结束后体系中存在的第一引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合靶标核酸的第一引物；The nucleic acid hybridization method according to claim 13 or 14, wherein the method further comprises: before performing step S2, using a ligase to ligate the first primers present in the system after the reaction of step S1 into a ring, and after the ring formation reaction is completed, performing an elution treatment with a washing solution to remove excess first primers that are not bound to the target nucleic acid; 任选地，所述方法进一步包括：在进行步骤S3之前，利用连接酶将步骤S2反应结束后体系中存在的第二引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合第一引物的第二引物；Optionally, the method further comprises: before performing step S3, using a ligase to ligate the second primer present in the system after the reaction in step S2 into a ring, and after the ring formation reaction is completed, performing an elution treatment using a washing solution to remove excess second primer that is not bound to the first primer; 任选地，所述方法进一步包括：在进行步骤S3之后，利用连接酶将步骤S3反应结束后体系中存在的第三引物连接成环，在成环反应结束之后，利用清洗液进行洗脱处理，以便去除多余的未结合第二引物的第三引物；Optionally, the method further comprises: after performing step S3, using a ligase to ligate the third primer present in the system after the reaction in step S3 into a ring, and after the ring formation reaction is completed, performing an elution treatment using a washing solution to remove excess third primer that is not bound to the second primer; 任选地，利用所述连接酶进行连接反应的反应温度为30-40℃，反应时间为5-30min。Optionally, the reaction temperature for the ligation reaction using the ligase is 30-40° C., and the reaction time is 5-30 min. 根据权利要求13-15中任一项所述的核酸杂交方法，其中，所述连接酶为T4连接酶；The nucleic acid hybridization method according to any one of claims 13 to 15, wherein the ligase is T4 ligase; 任选地，所述清洗液包括碳酸乙烯酯，所述碳酸乙烯酯在所述清洗液中的浓度为5-40％v/v；Optionally, the cleaning solution comprises ethylene carbonate, and the concentration of ethylene carbonate in the cleaning solution is 5-40% v/v; 任选地，所述清洗液进一步包括硫酸葡聚糖和Triton-X-100，其中，所述硫酸葡聚糖的浓度为5-20％，所述Triton-X-100的浓度为0.05-0.5％。Optionally, the cleaning solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%. 根据权利要求13-16中任一项所述的核酸杂交方法，其中，步骤S1中配对结合的反应温度为30-40℃，反应时间为30-120min；The nucleic acid hybridization method according to any one of claims 13 to 16, wherein the reaction temperature for pairing and binding in step S1 is 30-40° C., and the reaction time is 30-120 min; 任选地，步骤S2中配对结合的反应温度为30-40℃，反应时间为20-40min；Optionally, the reaction temperature for pairing in step S2 is 30-40°C, and the reaction time is 20-40 min; 任选地，步骤S3中配对结合的反应温度为30-40℃，反应时间为20-40min。Optionally, the reaction temperature for the pairing binding in step S3 is 30-40° C., and the reaction time is 20-40 min. 根据权利要求13-17中任一项所述的核酸杂交方法，其中，所述方法进一步包括：The nucleic acid hybridization method according to any one of claims 13 to 17, wherein the method further comprises: Ⅰ.在步骤S3之后，继续重复步骤S2-S3至少N轮，N≥1，N为整数；或者，Ⅰ. After step S3, continue to repeat steps S2-S3 for at least N rounds, where N≥1 and N is an integer; or, Ⅱ.在步骤S3之后，继续重复步骤S2-S3至少N’轮，N’≥0，N’为整数，且在最后一轮仅重复步骤S2。II. After step S3, continue to repeat steps S2-S3 for at least N' rounds, N'≥0, N' is an integer, and only repeat step S2 in the last round. 一种检测靶标核酸的方法，其包括：利用权利要求13-18中任一项所述的核酸杂交方 法获得靶标核酸、第一引物、第二引物、任选的第三引物组成的杂交体，利用第一检测探针和/或第二检测探针检测所述杂交体；A method for detecting a target nucleic acid, comprising: utilizing the nucleic acid hybridization method according to any one of claims 13 to 18 A method for obtaining a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer, and detecting the hybrid using a first detection probe and/or a second detection probe; 其中，所述第一检测探针包括与所述第二引物中的不与所述第一引物配对的区域的至少一部分配对结合的序列；wherein the first detection probe comprises a sequence that pairs and binds to at least a portion of the region of the second primer that does not pair with the first primer; 所述第二检测探针包括与所述第三引物中的不与所述第二引物配对的区域的至少一部分配对结合的序列；The second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that does not pair with the second primer; 所述第一检测探针和所述第二检测探针含有可检测信号的标记物。The first detection probe and the second detection probe contain a label capable of generating a detectable signal. 根据权利要求19所述的方法，其中，所述第一检测探针包括与所述第二引物中的第二区域的至少一部分配对结合的序列；The method of claim 19, wherein the first detection probe comprises a sequence that pairs and binds to at least a portion of the second region in the second primer; 任选地，所述第一检测探针包括与所述第二引物中的第七片段的至少一部分和第八片段的至少一部分配对结合的序列。Optionally, the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer. 根据权利要求19或20所述的方法，其中，所述第二检测探针包括与所述第三引物中的第三区域的至少一部分配对结合的序列；The method according to claim 19 or 20, wherein the second detection probe comprises a sequence that pairs and binds to at least a portion of the third region in the third primer; 任选地，所述第二检测探针包括与所述第三引物中的第三片段的至少一部分和第四片段的至少一部分配对结合的序列。Optionally, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer. 根据权利要求19-21中任一项所述的方法，其中，所述方法进一步包括：The method according to any one of claims 19 to 21, wherein the method further comprises: i.在步骤S2之后，以所述第一检测探针检测所述靶标核酸、第一引物以及第二引物组成的杂交体；或者i. After step S2, detecting the hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe; or ⅰi.在步骤S3之后继续重复步骤S2-S3至少N轮，以所述第二检测探针检测所述靶标核酸、第一引物、第二引物以及第三引物组成的杂交体，任选地，利用所述第一检测探针进行辅助检测，其中N为自然数；或者i. after step S3, continue to repeat steps S2-S3 for at least N rounds, detecting the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer with the second detection probe, optionally using the first detection probe for auxiliary detection, wherein N is a natural number; or ⅱi.在步骤S3之后继续重复步骤S2-S3至少N’轮，且在最后一轮仅重复步骤S2的情况，则以所述第一检测探针检测所述靶标核酸、第一引物、第二引物以及第三引物组成的杂交体，任选地，利用所述第二检测探针进行辅助检测，其中N’为正整数。ii. After step S3, continue to repeat steps S2-S3 for at least N' rounds, and in the case of repeating only step S2 in the last round, the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer is detected with the first detection probe, and optionally, the second detection probe is used for auxiliary detection, wherein N' is a positive integer. 根据权利要求19-22中任一项所述的方法，其中，所述第一、二检测探针的探针长度分别独立地为13-23nt。The method according to any one of claims 19 to 22, wherein the probe lengths of the first and second detection probes are independently 13-23 nt. 一种核酸、蛋白质联合检测的方法，其包括以下步骤：A method for combined detection of nucleic acids and proteins, comprising the following steps: 1)利用权利要求19-23中任一项所述的检测靶标核酸的方法对含有核酸的待测生物样本进行核酸检测；以及1) performing nucleic acid detection on a biological sample containing nucleic acid using the method for detecting a target nucleic acid according to any one of claims 19 to 23; and 2)对所述生物样本进行蛋白质荧光染色与成像。2) performing protein fluorescence staining and imaging on the biological sample. 根据权利要求24所述的方法，其中，所述蛋白质荧光染色包括利用抗体进行蛋白质多轮染色。The method according to claim 24, wherein the protein fluorescent staining comprises multiple rounds of protein staining using antibodies. 权利要求1-8中任一项所述的引物组合、权利要求9-12中任一项所述的套装在以下中的用途：Use of the primer combination according to any one of claims 1 to 8 and the kit according to any one of claims 9 to 12 in the following: a.核酸原位杂交；a. Nucleic acid in situ hybridization; b.核酸、蛋白质联合检测；b. Combined detection of nucleic acid and protein; c.组织图谱构建；c. Organizational map construction; d.确定组织中细胞分布；d. Determine the distribution of cells in tissues; e.数字病理成像和分析；e. Digital pathology imaging and analysis; f.生物标志物的筛选和发现。 f. Biomarker screening and discovery.
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