CN113151608B - PCR target sequence, primer and probe for detecting infectious SARS-CoV-2 and application - Google Patents
PCR target sequence, primer and probe for detecting infectious SARS-CoV-2 and application Download PDFInfo
- Publication number
- CN113151608B CN113151608B CN202110578905.7A CN202110578905A CN113151608B CN 113151608 B CN113151608 B CN 113151608B CN 202110578905 A CN202110578905 A CN 202110578905A CN 113151608 B CN113151608 B CN 113151608B
- Authority
- CN
- China
- Prior art keywords
- cov
- artificial sequence
- sars
- dna
- primer
- 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.)
- Active
Links
- 239000000523 sample Substances 0.000 title claims abstract description 117
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 54
- 230000002458 infectious effect Effects 0.000 title claims abstract description 51
- 241001678559 COVID-19 virus Species 0.000 title claims abstract 10
- 241000700605 Viruses Species 0.000 claims abstract description 37
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 33
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 31
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims description 27
- 238000011529 RT qPCR Methods 0.000 claims description 20
- 230000002779 inactivation Effects 0.000 abstract description 18
- 238000012360 testing method Methods 0.000 abstract description 17
- 108090000565 Capsid Proteins Proteins 0.000 abstract description 10
- 102100023321 Ceruloplasmin Human genes 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 230000003612 virological effect Effects 0.000 abstract description 4
- 239000002245 particle Substances 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 241000315672 SARS coronavirus Species 0.000 description 65
- 108020004414 DNA Proteins 0.000 description 62
- 238000011534 incubation Methods 0.000 description 22
- 230000003321 amplification Effects 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 17
- 238000003199 nucleic acid amplification method Methods 0.000 description 17
- 238000006303 photolysis reaction Methods 0.000 description 13
- 230000015843 photosynthesis, light reaction Effects 0.000 description 13
- 241000711573 Coronaviridae Species 0.000 description 12
- 239000013068 control sample Substances 0.000 description 11
- 239000013504 Triton X-100 Substances 0.000 description 9
- 229920004890 Triton X-100 Polymers 0.000 description 9
- 238000000137 annealing Methods 0.000 description 9
- 239000000645 desinfectant Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 102000016911 Deoxyribonucleases Human genes 0.000 description 6
- 108010053770 Deoxyribonucleases Proteins 0.000 description 6
- 102000006382 Ribonucleases Human genes 0.000 description 6
- 108010083644 Ribonucleases Proteins 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000008055 phosphate buffer solution Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 208000025721 COVID-19 Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 229960002233 benzalkonium bromide Drugs 0.000 description 2
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229920002113 octoxynol Polymers 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101000595467 Homo sapiens T-complex protein 1 subunit gamma Proteins 0.000 description 1
- 102100036049 T-complex protein 1 subunit gamma Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- VCWNAJUHTLWQQT-YRGDBDHISA-N mpma Chemical compound C([C@@]1(C(=O)C(C)=CC1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)OC)C(CO)=C[C@H]1C1[C@]2(OC(C)=O)C1(C)C VCWNAJUHTLWQQT-YRGDBDHISA-N 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 238000011369 optimal treatment Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
Images
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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及SARS‑CoV‑2核酸检测领域，更具体地，涉及检测具有感染性SARS‑CoV‑2的PCR靶序列、引物和探针及应用。本发明提供的检测具有感染性SARS‑CoV‑2的PCR靶序列、引物和探针可以很好的区分SARS‑CoV‑2的感染性，同时具有灵敏度高、重复性好的特点。与常规感染性SARS‑CoV‑2检测试验相比，本发明极大缩短了感染性SARS‑CoV‑2检测的试验周期，降低了对科研条件和设备的要求；有效区分了病毒衣壳蛋白损伤导致病毒失活的核酸与和感染性病毒粒子，提高了检测的准确性，减少了实际检测中出现的假阳性现象。The invention relates to the field of SARS-CoV-2 nucleic acid detection, and more particularly, to PCR target sequences, primers and probes for detecting infectious SARS-CoV-2, and applications. The PCR target sequences, primers and probes for detecting infectious SARS-CoV-2 provided by the present invention can well distinguish the infectivity of SARS-CoV-2, and have the characteristics of high sensitivity and good repeatability. Compared with the conventional infectious SARS-CoV-2 detection test, the present invention greatly shortens the test period for infectious SARS-CoV-2 detection, reduces the requirements for scientific research conditions and equipment; effectively distinguishes the damage of the viral capsid protein The nucleic acid and infectious virus particles that cause virus inactivation improve the accuracy of detection and reduce false positives in actual detection.
Description
Technical Field
The invention relates to the field of SARS-CoV-2 nucleic acid detection, in particular to a PCR target sequence, a primer and a probe for detecting infectious SARS-CoV-2 and application thereof.
Background
Pneumonia (Corona Virus Disease 2019, CoVID-19 for short) infected by severe acute respiratory syndrome coronavirus type 2 (SAR S-CoV-2, new coronavirus for short) is an infectious Disease transmitted through respiratory tract.
Currently, the detection of novel coronavirus nucleic acid is a gold standard for determining new coronary pneumonia, screening asymptomatic infectors, investigating cold chain food and environmental pollution, and is a key point for epidemic prevention and control. At present, the nucleic acid detection means for the novel coronavirus is mainly qRT-PCR method, and the method plays an important role in the detection of the novel coronavirus due to higher specificity and sensitivity. However, the method can only detect whether the nucleic acid sequence of the virus exists in the sample, and cannot give effective indication whether the virus in the sample has capsid protein integrity, infectivity and the like. At present, a large number of novel coronavirus detection nucleic acid positive events in places such as hospital handles, air and cold chain transportation are reported at home and abroad. More detailed studies have found that the positive nucleic acid detection is mostly due to "false positive" caused by inactivation measures such as high temperature and disinfectant in these locations. For the novel coronavirus, whether the virus in a sample still has infectivity after inactivation measures such as heat inactivation and disinfectant sterilization are adopted, and a classical virus culture and separation method is also needed for determining. Because the novel coronavirus is a new highly pathogenic pathogen, the conventional traditional culture and serology and other common clinical methods need to be carried out in a P3-grade laboratory under the condition of biosafety protection and are difficult to detect the pathogen in a short time, so that the infectivity detection of the novel coronavirus through isolated culture is not feasible for basic units or cold chain transportation places, and great inconvenience is brought to the actual prevention and control of the novel coronavirus, such as determination of infection ways, whether the environment is still provided with infectious viruses after disinfection treatment in the places such as hospitals and cold chain transportation. Therefore, there is an urgent need to develop a rapid and simple alternative technique for detecting infectious novel coronaviruses.
Azidopropidium bromide (PMA) is a light-sensitive nucleic acid dye with high affinity for DNA or RNA, and PMA cannot penetrate the intact viral capsid proteins and can only selectively bind to nucleic acids exposed after the viral capsid proteins have been damaged. The integrity of the virus capsid protein is a mark for judging the virus activity, and the virus is mostly sterilized by high-temperature inactivation or chemical disinfectant inactivation in the daily industrial production, hospitals, laboratories, cold chain transportation and other environments, and the inactivation methods and chemical preparations mainly inactivate the virus by destroying the virus capsid protein structure. PMA can penetrate damaged virus capsid protein to be combined with virus nucleic acid, after incubation and photolysis under certain conditions, PMA can be stably crosslinked with exposed nucleic acid to prevent subsequent PCR reaction of nucleic acid, thereby removing interference (false positive) caused by nucleic acid exposed by inactivated virus and achieving the purpose of detecting whether a sample contains infectious virus. In addition, the low-concentration nonionic surfactant Triton X-100 can be used as an enhancer for PMA pretreatment, and the capability of PMA in detecting the false positive of infectious viruses is improved by selectively reducing the permeability of damaged capsid proteins of inactivated viruses to combine exposed nucleic acid with more PMA.
PMA-qPCR has been used well in detecting infectious bacteria and viruses in food, environment, water and other samples, but has not been reported in detecting infectious SARS-CoV-2. The efficiency of PMA in inhibiting the PCR amplification of capsid protein-impaired virus depends on the length of the amplified target fragment and the position of the target gene, in addition to PMA incubation conditions, selection of an enhancer, incubation temperature, photolysis time and the like. When the PMA enters the virus with damaged capsid protein, the PMA is combined with virus nucleic acid in a certain stoichiometric ratio, theoretically, the target gene is modified by the PMA to inhibit the PCR amplification of the target fragment, therefore, the probability of combination with the PMA is increased by a longer target gene amplification fragment, thereby improving the inhibition effect of the PMA, but the amplification efficiency and sensitivity of PCR reaction can be reduced by a longer amplification fragment. In addition, different regions and different positions in the viral genome have different binding efficiencies to PMA due to their different GC contents, base bias, and conformational characteristics. In the test of the effect of inhibiting the bacteria killed by the PMA of the escherichia coli, target gene segments with the same length but different positions are selected to obtain different PMA inhibiting effects. Therefore, the method for detecting infectious SARS-CoV-2 is important for the development of the technical method for balancing the length of target fragments and the PMA inhibition efficiency and screening out proper PMA binding regions in SARS-CoV-2 genome.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting a target point sequence based on a PMA-qPCR method in preparing a detection kit for distinguishing nucleic acid positive from virus positive of SARS-CoV-2, wherein the nucleotide sequence of the target point sequence is shown as SEQ ID NO. 61.
In order to achieve the purpose, the invention adopts the following technical measures:
the application of a reagent for detecting a target sequence based on a PMA-qPCR method in preparing a detection kit for distinguishing nucleic acid positive and virus positive of SARS-CoV-2 is disclosed, wherein the nucleotide sequence of the target sequence is shown as SEQ ID NO. 61. In the above application, preferably, the reagent for detecting the target sequence based on the PMA-qPCR method is a primer and a probe, and the primer specifically is: TTCACACAATCGACGGTTCATCC and GTACCTGTCTCTTCCGAAACGAAT, the probes are: ACGACGACTACTAGCGTGCCTT, the probe carries a fluorophore as is conventional in the art.
In the above application, preferably, in the application process of the kit, the pretreatment mode of the sample to be detected is as follows:
(1) placing a sample to be detected in an EP tube without DNase/RNase enzyme for centrifugation, and collecting a supernatant;
(2) putting 180 μ l of the supernatant into an EP tube without DNase and RNase, adding 10 μ l of PMA and 10 μ l of Triton X-100 in a dark place, mixing uniformly, and performing vortex incubation at 37 ℃ in a dark place for 30 min; the final concentration of PMA is 100 mu M; the final concentration of Triton X-100 is 0.5%.
(3) The samples were transferred to DNase/RNase enzyme-free binding EP tubes and PMA-Lite was usedTMAnd (3) photolyzing the sample for 20min by using an LED photolysis instrument.
In the above application, preferably, the system of the kit in the application process is:
Compared with the prior art, the invention has the following advantages:
the PCR primer and probe for detecting SARS-CoV-2 provided by the invention can well distinguish the infectivity of SARS-CoV-2, and simultaneously has the characteristics of high sensitivity and good repeatability. The primer probe and the detection method are applied to the multiplex qRT-PCR kit for detecting infectious SARS-CoV-2, and all experiments can be completed within 2-3 hours. Compared with the conventional infectious SAR S-CoV-2 detection test, the invention greatly shortens the test period of infectious SARS-CoV-2 detection and reduces the requirements on scientific research conditions and equipment; effectively distinguishes nucleic acid and infectious virus particles which cause virus inactivation due to virus capsid protein damage, improves the detection accuracy and reduces the false positive phenomenon in the actual detection. The kit can be used for detecting samples in public environments, production areas, office places, food and food surfaces and the like, is suitable for hospitals, laboratories, basic prevention and control units, cold chain transportation places and the like, and has important reference significance and good application prospect for determining the actual prevention and control of SARS-CoV-2, such as determining infection routes, whether infectious viruses exist in the environments after the disinfection treatment in the places such as hospitals, cold chain transportation and the like.
Drawings
FIG. 1 illustrates qRT-PCR amplification curves and standard curves for detecting infectious SARS-CoV-2 primer probe sets;
wherein A is the amplification curve chart of the primer probe group for detecting infectious SARS-CoV-2;
b is a standard curve chart for detecting infectious SARS-CoV-2 primer probe set.
FIG. 2 shows the qRT-PCR amplification curve and the standard curve of the Chinese CDC-N primer probe set.
Wherein A is an amplification curve chart of the Chinese CDC-N primer probe group for detecting infectious SARS-CoV-2;
b is the standard curve chart of the Chinese CDC-N primer probe set for detecting infectious SARS-CoV-2.
Detailed Description
For a better understanding of the present disclosure, the following further description is provided in conjunction with the specific embodiments, but the present disclosure is not limited to the following examples. Unless otherwise specified, the test methods and conditions in the examples of the present invention are conventional methods. The technical scheme of the invention is a conventional scheme in the field if no special description exists; the reagents or materials are commercially available, unless otherwise specified. All tests related to severe acute respiratory syndrome coronavirus (SARS-CoV-2) as SARS-CoV-2 strain (ZY38-1) in the invention are completed in biosafety tertiary laboratory (ABSL 3). The following specific examples further illustrate the invention:
the SARS-CoV-2 strain (ZY38-1) has been delivered to the China Center for Type Culture Collection (CCTCC) at 04.01.2021, and is classified and named: SARS-CoV-2/ZY38-1, accession number: CCTCC NO: V202107, address: wuhan university in Wuhan, China.
Example 1:
screening and optimizing primers and probes for detecting infectious SARS-CoV-2:
1.1 design of infectious SARS-CoV-2 primers and probes
Combining 1-15 (SEQ ID NO. 1-SEQ ID NO.45) primer probe sets according to SARS-CoV-2(ZY38-1) gene, and referencing and combining the primer probe sets for detecting SARS-CoV-2 issued by the Chinese CDC; china CDC-ORF1ab and China CDC-N (SEQ ID NO. 46-SEQ ID NO.51), and U.S. CDC-N1-N3 (SEQ ID NO. 52-SEQ ID NO.60) as primer probe sets for detecting SARS-CoV-2 issued by U.S. CDC were referenced and synthesized. The sequences of the primers and the fluorescent probe are as follows (the primers and the probe are synthesized by Shanghai Biotech Co., Ltd.):
TABLE 1 SARS-CoV-2 primer, Probe sequences
1.2qRT-PCR reaction system and reaction conditions:
reaction system: enzyme Mix 2 mul, MP buffer 12 mul, upstream and downstream primer final concentration of 400-1000 pM each, probe final concentration of 200-400 pM each, template 2 mul, sterile water make up to 25 mul.
Reaction conditions are as follows: reverse transcription is carried out for 10min at 50 ℃, then pre-denaturation is carried out for 5min at 95 ℃, and then circulation is carried out for 45 times; the procedure for each cycle was: and (3) denaturation at 95 ℃ for 5sec, annealing at 58-62 ℃ for 30sec, collecting signals corresponding to the fluorescent channels of the probes, and finishing the reaction after the circulation is finished.
Wherein, the positive control (standard substance) is a synthesized virus nucleic acid sequence plasmid containing 1.1 primer amplified fragments, and the negative control is sterile water without RNA. The CT value of the positive control is less than 40, a typical amplification curve appears, the negative control has no C T value and no typical amplification curve indicates that the experimental result is established, the data listed below are all experimental result establishment data, and the CT value data of the positive control and the negative control are not repeated.
1.3 screening of infectious SARS-CoV-2 primers and probes
After being diluted, the SARS-CoV-2 infected vero cell virus harvest liquid is inactivated by using a novel coronavirus inactivation condition (56 ℃ heat inactivation for 30min) recommended by the world health organization and the Chinese disease control center, the inactivated sample is cultured for 7 days, and the negative side of a plaque test can be used as an inactivation completion sample to carry out the next test:
transferring two test samples with the same volume after inactivation is finished, adding PMA (final concentration is 100 mu M) and Triton X (final concentration is 0.5%) into one test sample, performing vortex incubation at room temperature for 20min in dark, and after incubation is finished, using PMA-LiteTMPerforming photolysis on the sample for 15min by using an LED photolysis instrument to serve as a pretreatment sample to be detected; and adding an equal amount of PBS into the other part of the sample to be detected, and incubating and photolyzing the other part of the sample to be detected and the pretreated sample to be detected under the same conditions to be used as a control sample to be detected. Adopting a full-automatic nucleic acid extractor for extracting PMA pretreatment (pretreatment sample to be detected) and P-freeNucleic acid of a sample (a control sample to be detected) is pretreated by MA, and by taking the nucleic acid as a template, qRT-PCR is carried out by using 1.1 synthesized primers and probes and 1.2 reaction systems and conditions (wherein the final concentration of the primers is 200pM, the final concentration of the probes is 400pM, and the annealing temperature is 58 ℃), and infectious SARS-CoV-2 primers and probes are screened.
TABLE 2 screening of infectious SARS-CoV-2 primer probes
As shown in Table 2, the CT values of the pre-treated samples to be tested of different primer probe sets are all increased compared with the control samples to be tested, and the larger the Delta CT value (CT value of the pre-treated sample to be tested-CT value of the control sample to be tested) is, the higher the binding efficiency of the amplified fragment of the primer probe set and PMA is, the stronger the effect on distinguishing infectious SARS-CoV-2 is. Wherein, the primer probe group 11(SEQ ID NO. 31-33) has the maximum delta CT value (the target sequence amplified by the primer group is shown in SEQ ID NO. 61), and is most suitable for detecting the positive (infectious) index of SARS-CoV-2 virus. In addition, the Chinese CDC-N primer probe group is a specific primer probe for detecting SARS-CoV-2, and simultaneously, the CT of a control sample to be detected is the lowest, and the detection has higher sensitivity, so the primer probe group is selected as a nucleic acid positive index in the method for detecting infectious SARS-CoV-2.
1.4qRT-PCR detection of infectious SARS-CoV-2 primers, Probe concentration and annealing temperature optimization
In order to obtain the optimal addition amount of the primers and the probes, the use concentrations and annealing temperatures of the primer probe set and the Chinese CDC-N primer probe set obtained in example 1.3 were optimized by using a 1.2qRT-PCR reaction system and reaction conditions, each ratio was repeated three times, and the Ct values under different primer and probe concentration conditions were measured as shown in tables 3 to 4.
TABLE 3 detection of infectious SARS-CoV-2 primer, Probe concentration, annealing temperature optimization
TABLE 4 optimization of primer, probe concentration and annealing temperature for Chinese CDC-N
As can be seen from the above table, the final concentration of the primers is 800pM, the final concentration of the probes is 400pM, and the Ct value of the amplification result is the lowest at the annealing temperature of 58 ℃, so that the concentration ratio (i.e., the final concentrations of the upstream and downstream primers are 800pM, and the final concentration of the probes is 400pM) is selected as the final concentration of the subsequent reaction.
1.5 amplification curve, standard curve and sensitivity:
the plasmid standard substance is diluted with sterilized water in 10 times gradient to obtain template with final dilution concentration of 2.5 × 108～2.5×100The copies/mu l standard substance is tested by adopting an optimal qRT-PCR reaction system and reaction conditions, and a standard curve equation of the logarithm value and the Ct value of the gene copy number is constructed. The lowest detection limit was determined using a negative control method (i.e., negative controls were tested multiple times, with 2 times the maximum test value of the negative controls being used as the detection limit).
The amplification curves of the primer probe group for detecting infectious SARS-CoV-2 and the primer probe group for Chinese CDC-N are shown in A in FIG. 1 and A in FIG. 2, and the results show a typical amplification curve chart and have good linear relationship; correlation coefficient R of standard curve20.9985 and 0.9971 respectively, and the amplification efficiency is 105.58% and 102.91% respectively, which indicates that the multiplex fluorescence quantification using the two pairs of primer probe sets has no cross influence and is feasible for detecting infectious SARS-CoV-2; the curve equations are Y-3.195X +44.76 (see B in fig. 1) and Y-3.254X +43.85 (see B in fig. 2), respectively, the lowest detection limit is 43.089copies/μ l and 35.127copies/μ l, respectively, and the coefficient of variation CV is 0.837% and 0.843%, respectively, indicating good sensitivity and reproducibility.
Example 2:
optimization of PMA-Triton X-100-qRT-PCR method for detecting infectious SARS-CoV-2
2.1PMA use concentration optimization
Respectively transferring test samples of inactivated samples in equal volume, adding PMA (final concentration of 0, 5, 50, 100, 150, 200, 250 μ M) with different concentrations, performing vortex incubation at room temperature for 20min in dark, and after incubation, using PMA-LiteTMAnd (3) photolyzing the sample for 15min by using an LED photolysis instrument. Nucleic acid is extracted by adopting a Tiangen full-automatic nucleic acid extractor, and qRT-PCR is carried out by taking the nucleic acid as a template. And comprehensively considering the use effect and the use cost, and selecting the lowest PMA concentration with larger delta Ct difference or Ct more than 40 of a sample to be processed without a typical amplification curve as the optimal PMA working concentration. As a result, the optimum working concentration of PMA was 100. mu.M, as shown in Table 5.
TABLE 5 optimal PMA use concentration exploration
| Final concentration of PMA (μ M) | Mean of CT values. + -. standard deviation | △ |
| 0 | 19.308±0.011 | 0.000 |
| 5 | 20.04±0.155 | 0.732 |
| 10 | 20.411±0.128 | 1.103 |
| 50 | 20.514±0.052 | 1.206 |
| 100 | 21.247±0.19 | 1.939 |
| 150 | 21.198±0.038 | 1.890 |
| 200 | 21.244±0.2 | 1.936 |
| 250 | 21.289±0.21 | 1.981 |
2.2PMA incubation conditions, incubation temperature, incubation time, photolysis time optimization
Test samples of inactivated samples were removed in equal volumes, PMA and TritonX-100 were added to the samples at final concentrations of (0. mu. MPMA + 0% Triton X-100; 100. mu.M PMA +0Triton X-100; 100. mu.M PMA + 0.5% Triton X-100), protected from light, incubated at 4, 25 and 37 ℃ for 5, 10 and 30min, respectively, followed by photolysis for 2, 12 and 20min, respectively. Nucleic acid is extracted by adopting a Tiangen full-automatic nucleic acid extractor, and qRT-PCR is carried out by taking the nucleic acid as a template. The incubation conditions with the largest delta Ct difference or the PMA pretreatment group Ct > 40 and no typical amplification curve were selected, and the incubation temperature, incubation time and photolysis time were used as the optimal treatment conditions. The results are shown in tables 6 to 9, and the optimal incubation conditions were 100. mu.M PMA + 0.5% Triton X-100; the optimal incubation temperature is 37 ℃; the optimal incubation time is 30 min; the optimal photolysis time is 20 min.
Table 6 exploration of optimal PMA incubation conditions
TABLE 7 optimal PMA incubation temperature exploration
TABLE 8 optimal PMA incubation time exploration
TABLE 9 optimal PMA photolysis time exploration
Example 3:
application of multiplex qRT-PCR kit for detecting SARS-CoV-2 infectivity
In this example, a food package surface sample (negative in plaque test verification as a group completed with inactivation) treated by a high-temperature alcohol disinfectant (75% ethanol), a chlorine-containing disinfectant (84 disinfectant) and a quaternary ammonium salt disinfectant (benzalkonium bromide) is simulated by referring to a specific site disinfection technical scheme (national defense office health control function [2020] 156) and a food safety national standard food cold chain logistics sanitary standard (GB31605-2020), and a virus liquid positive in SARS-CoV-2 plaque test is taken as a group not yet inactivated. The optimal primer, probe and treatment method for detecting infectious SARS-CoV-2 obtained in examples 1-2 are applied to the multiple qRT-PCR kit for detecting infectious SARS-CoV-2.
A sample collection step:
refer to the section for sample Collection in methods for sampling and detecting New coronavirus in food and food packaging surfaces (T/CAQI 159-.
A detection step:
(1) taking a sample to be detected, equally dividing the sample into 2 parts, pretreating one part of the sample by using PMA-Triton X and the sample to be detected as a pretreatment sample to be detected, and adding an equal amount of PBS (phosphate buffer solution) into the other part of the sample to be detected as a control sample to be detected;
(2) respectively extracting viruses RN A of a pretreatment sample to be detected and a control sample to be detected by using a Tiangen full-automatic nucleic acid extractor;
(3) and respectively carrying out multiplex qRT-PCR by taking the RNA of the pretreatment sample to be detected and the RNA of the control sample to be detected as templates.
The reaction system is as follows: enzyme Mix 2 mul, MP buffer 12 mul, detecting infectious SARS-CoV-2 upstream and downstream primers (SEQ ID NO. 31-32) and Chinese CDC-N upstream and downstream primers (SEQ ID NO. 49-50), the final concentration of the primers is 800pM, detecting infectious SARS-CoV-2 probe (SEQ ID NO.33) and Chinese CDC-N probe (SEQ ID NO.51) are 400pM each, the template is 2 mul, and sterilized water is supplemented to 25 mul.
The reaction conditions are as follows: reverse transcription is carried out for 10min at 50 ℃, then pre-denaturation is carried out for 5min at 95 ℃, and then circulation is carried out for 45 times; the procedure for each cycle was: denaturation at 95 ℃ for 5sec, annealing at 58 ℃ for 30sec, collecting the signal corresponding to the fluorescent channel of the probe, and terminating the reaction after the end of the cycle.
Wherein, the positive control is a synthesized virus nucleic acid sequence plasmid containing a target gene segment, and the negative control is sterile water containing no RNA.
(4) Using CT value of the control sample to be detected obtained by Chinese CDC-N primer probe group as SARS-CoV-2 nucleic acid (positive/negative) index; taking the difference (delta Ct) of the Ct values of the pretreatment sample to be detected and the control sample to be detected, which are obtained by detecting the infectious SARS-CoV-2 primer probe group, as the positive/negative (infectious) index of SARS-CoV-2 virus: the delta Ct is more than 6 or the Ct value of the pre-treated sample to be detected is more than 40, and no obvious amplification curve exists, namely the virus is SARS-CoV-2 virus negative (without infectivity), and the delta Ct is less than or equal to 6, namely the virus is SARS-CoV-2 virus positive (with infectivity).
Further: the specific steps of the pretreatment of the sample to be detected comprise the following steps:
(1) placing a sample to be detected in an EP tube without DNase/RNase enzyme for centrifugation, and collecting a supernatant;
(2) putting 180 μ l of the supernatant into an EP tube without DNase and RNase, adding 10 μ l of PM A and 10 μ l of Triton X-100 in a dark place, mixing uniformly, and performing vortex incubation at 37 ℃ in a dark place for 30 min; the final concentration of PMA is 100 mu M; the final concentration of Triton X-100 is 0.5%.
(3) The samples were transferred to DNase/RNase enzyme-free binding EP tubes and PMA-Lite was usedTMAnd (3) photolyzing the sample for 20min by using an LED photolysis instrument.
And (3) performing incubation and photolysis on the control sample to be detected and the pretreatment sample to be detected under the same conditions, but adding PMA and Triton X-100 into the control sample to be detected, and only adding PBS with the same volume.
The results of applying the multiple qRT-PCR kit for detecting infectious SARS-CoV-2 to food package surface samples treated by different inactivation methods are shown in Table 10.
In addition, in this example, the effect of the conventional primer-probe set on the detection of infectious SAR S-CoV-2 was examined by using the Chinese CDC-ORF primer probe set (SEQ ID NO. 46-48), the Chinese CDC-N primer probe set (SEQ ID NO. 49-51), the U.S. CDC-N1 primer probe set (SEQ ID NO. 52-54), the U.S. CDC-N2 primer probe set (SEQ ID NO. 55-57) and the U.S. CDC-N3 primer probe set (SEQ ID NO. 58-60) as positive/negative (infectivity) indicators of SARS-CoV-2 virus. The procedure of the application of the multiplex qRT-PCR kit for detecting SARS-CoV-2 infectivity in example 3 was the same except that the primer probe set was changed, the reaction system and conditions were referred to the recommended concentration of primer probe and annealing temperature published in China and U.S. CDC.
The results of detecting the food packaging surface samples treated by different inactivation methods by the primer probe set in Zhongmei are shown in Table 11.
TABLE 10 detection of infectious SARS-CoV-2 multiplex qRT-PCR kit applied to food package surface samples treated by different inactivation methods
Table 11 results of detection of food packaging surface samples treated by different inactivation methods by using American CDC primer probe set
The data in Table 10 show that all the nucleic acid indicators were positive. Wherein, the infectivity index delta CT of a sample (completely inactivated sample) subjected to high temperature, 84, benzalkonium bromide and alcohol inactivation is more than 6, or the CT value of a pretreatment sample to be detected is more than 40, and no typical amplification curve exists, which indicates that the sample has no infectivity. The infectivity index delta CT of the inactivated sample is less than 6, which shows that the sample has infectivity, the detection result of the primer probe for detecting the infectivity SARS-CoV-2 is consistent with the result of the plaque test, and the kit of the invention can achieve the purpose of quickly detecting the infectivity SARS-CoV-2.
The data in Table 11 show that the Delta CT obtained by detecting samples inactivated by different methods by using the primer probe set in China and American CDC as the infectivity index is small, and the conventional SARS-CoV-2 primer probe set has poor effect on detecting the infectivity of the virus and cannot achieve the purpose of distinguishing the infectious SARS-CoV-2.
The kit can complete the detection of SARS-CoV-2 infectivity within 2-3 h, has the advantages of rapidness, simplicity, high efficiency and the like, and can be used for the primary screening of SARS-CoV-2 infectivity rapid detection.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> PCR target sequence, primer and probe for detecting infectious SARS-CoV-2 and application
<160> 61
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tagtttagct gccacagtac gtc 23
<210> 2
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cagcaaaagc acagaaagat aatacagt 28
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aattggcagg cacttctgtt gcat 24
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aatacctaca acttgtgcta atgacc 26
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgtgctgact ctatcattat tggt 24
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gataatttct tttggggctt ttagagg 27
<210> 9
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ttctgtacaa tccctttgag tgcgtgac 28
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctgacataga agttactggc gat 23
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cgcattaata tgacgcgcac t 21
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aaacatgaca ccccgtgacc t 21
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gttttatgag gctatgtaca caccg 25
<210> 14
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tttatgtgat gttgatatga catggtcgt 29
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agccccaaca gcctgtaaga ct 22
<210> 16
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ttatcctaaa tgtgatagag ccatgcc 27
<210> 17
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cacctggttt aacatatagt gaaccg 26
<210> 18
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acgtgttgta gcttgtcaca ccgt 24
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tactgttttg ccacctttgc tc 22
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gcaccaaagg tccaaccag 19
<210> 21
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ctgcactgtt agcgggtaca atcact 26
<210> 22
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ttcatcaaac aatatggtga ttgcct 26
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gcaccaaagg tccaaccag 19
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tctgcactgt tagcgggtac aatcact 27
<210> 25
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cgcgctactg caacgat 17
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gttgcaaaca aagtgaacac cc 22
<210> 27
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
cgatacaagc ctcactccct ttcggat 27
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gttgcaaaca aagtgaacac cc 22
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgcaacgcca acaataagcc atccg 25
<210> 31
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ttcacacaat cgacggttca tcc 23
<210> 32
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gtacctgtct cttccgaaac gaat 24
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
acgacgacta ctagcgtgcc tt 22
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
cgtggacatc ttcgtattgc tg 22
<210> 35
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gccaatcctg tagcgact 18
<210> 36
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cactgttgct acatcacgaa cgctt 25
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
<210> 38
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ccaatttgta ataagaaagc gttcgt 26
<210> 39
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
acatcaagga cctgcctaaa gaaatcact 29
<210> 40
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cctttaattg aattgtgcgt ggat 24
<210> 41
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gatgaaatct aaaacaacac gaacgtc 27
<210> 42
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
aacgaacaac gcactacaag actaccc 27
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
aaaacctgag tcacctgcta c 21
<210> 45
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
acatcaagga cctgcctaaa gaaatcact 29
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ccctgtgggt tttacactta a 21
<210> 47
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
acgattgtgc atcagctga 19
<210> 48
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ccgtctgcgg tatgtggaaa ggttatg 27
<210> 49
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ggggaacttc tcctgctaga at 22
<210> 50
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cagacatttt gctctcaagc tg 22
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
<210> 53
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tctggttact gccagttgaa tctg 24
<210> 54
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
accccgcatt acgtttggtg gacc 24
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
<210> 56
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gcgcgacatt ccgaagaa 18
<210> 57
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
acaatttgcc cccagcgctt cag 23
<210> 58
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gggagccttg aatacaccaa aa 22
<210> 59
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tgtagcacga ttgcagcatt g 21
<210> 60
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
aycacattgg cacccgcaat cctg 24
<210> 61
<211> 148
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ttcacacaat cgacggttca tccggagttg ttaatccagt aatggaacca atttatgatg 60
aaccgacgac gactactagc gtgcctttgt aagcacaagc tgatgagtac gaacttatgt 120
actcattcgt ttcggaagag acaggtac 148
Claims (1)
1. The application of primer combination based on PMA-qPCR method in preparing detection kit for distinguishing nucleic acid positivity of SARS-CoV-2 from virus positivity, the described primer combination is primer and probe for detecting infectious SARS-CoV-2 and primer and probe for detecting Chinese CDC-N;
the primer for detecting infectious SARS-CoV-2 is as follows: TTCACACAATCGACGGTTCATCC and GTACCTGTCTCTTCCGAAACGAAT, the probes are: ACGACGACTACTAGCGTGCCTT, respectively;
the primers of the Chinese CDC-N are GGGGAACTTCTCCTGCTAGAAT and CAGACATTTTGCTCTCAAGCTG, and the probes are as follows: TTGCTGCTGCTTGACAGATT are provided.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110578905.7A CN113151608B (en) | 2021-05-26 | 2021-05-26 | PCR target sequence, primer and probe for detecting infectious SARS-CoV-2 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110578905.7A CN113151608B (en) | 2021-05-26 | 2021-05-26 | PCR target sequence, primer and probe for detecting infectious SARS-CoV-2 and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113151608A CN113151608A (en) | 2021-07-23 |
| CN113151608B true CN113151608B (en) | 2021-11-05 |
Family
ID=76877603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110578905.7A Active CN113151608B (en) | 2021-05-26 | 2021-05-26 | PCR target sequence, primer and probe for detecting infectious SARS-CoV-2 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113151608B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113832262A (en) * | 2021-11-10 | 2021-12-24 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Oligonucleotide for detecting infectivity of novel coronavirus, kit containing oligonucleotide and application |
| CN119082376A (en) * | 2024-11-07 | 2024-12-06 | 南京英诺唯真生物科技有限公司 | A kit and method for detecting novel coronavirus |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982943A (en) * | 2020-03-03 | 2020-04-10 | 中山大学达安基因股份有限公司 | Novel coronavirus RT-PCR detection method and kit |
| CN111057797A (en) * | 2020-01-19 | 2020-04-24 | 华中科技大学同济医学院附属同济医院 | Novel coronavirus 2019-nCoV real-time quantitative PCR detection primers and probes, kits and methods |
| CN111139242A (en) * | 2020-04-03 | 2020-05-12 | 苏州吉玛基因股份有限公司 | Small interfering nucleic acid, composition and application |
| CN111139241A (en) * | 2020-04-03 | 2020-05-12 | 苏州吉玛基因股份有限公司 | Small interfering nucleic acid for inhibiting novel coronavirus, composition and application |
| US10815539B1 (en) * | 2020-03-31 | 2020-10-27 | Diasorin S.P.A. | Assays for the detection of SARS-CoV-2 |
-
2021
- 2021-05-26 CN CN202110578905.7A patent/CN113151608B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057797A (en) * | 2020-01-19 | 2020-04-24 | 华中科技大学同济医学院附属同济医院 | Novel coronavirus 2019-nCoV real-time quantitative PCR detection primers and probes, kits and methods |
| CN110982943A (en) * | 2020-03-03 | 2020-04-10 | 中山大学达安基因股份有限公司 | Novel coronavirus RT-PCR detection method and kit |
| US10815539B1 (en) * | 2020-03-31 | 2020-10-27 | Diasorin S.P.A. | Assays for the detection of SARS-CoV-2 |
| CN111139242A (en) * | 2020-04-03 | 2020-05-12 | 苏州吉玛基因股份有限公司 | Small interfering nucleic acid, composition and application |
| CN111139241A (en) * | 2020-04-03 | 2020-05-12 | 苏州吉玛基因股份有限公司 | Small interfering nucleic acid for inhibiting novel coronavirus, composition and application |
Non-Patent Citations (2)
| Title |
|---|
| Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications–A systematic review;Mats Leifels等;《Water Research X》;20201209;第11卷;100080 * |
| 叠氮溴化丙锭结合qPCR检测与区分活菌和死菌的研究进展;陶怡君等;《微生物学免疫学进展》;20201231;第48卷(第6期);第63-68页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113151608A (en) | 2021-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113502352B (en) | EMA-ddPCR primer and probe for detecting infectious ASFV and application | |
| CN111321234B (en) | Method for detecting microorganisms based on CRISPR-Cas13a system and application | |
| Monpoeho et al. | Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid | |
| CN110894556A (en) | PCR primers and probes, kits and detection methods for detecting African swine fever virus infectivity | |
| Zhou et al. | Rapid detection of monkeypox virus by multiple cross displacement amplification combined with nanoparticle‐based biosensor platform | |
| CN114934102B (en) | Primer group and kit for simultaneously detecting multiple respiratory tract pathogen nucleic acids based on icosaPCR | |
| Ravan et al. | Loop region-specific oligonucleotide probes for loop-mediated isothermal amplification–enzyme-linked immunosorbent assay truly minimize the instrument needed for detection process | |
| CN113151608B (en) | PCR target sequence, primer and probe for detecting infectious SARS-CoV-2 and application | |
| CN108410957B (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and detection kit for detecting mycoplasma genitalium | |
| CN113652505B (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| Lau et al. | Detection of foot-and-mouth disease virus by nucleic acid sequence-based amplification (NASBA) | |
| Tran et al. | A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care | |
| CN113718045A (en) | DNA fragment, primer, probe and kit for detecting 4 kinds of Bordetella pertussis and specifically detecting Bordetella pertussis and application | |
| CN112280879A (en) | RPA primer and kit for rapidly detecting citrus yellow shoot Asian species, detection method and application thereof | |
| CN101177714A (en) | PCR detection method of Listeria monocytogenes with added internal standard | |
| CN108411014A (en) | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida | |
| CN104611420A (en) | Tubercle bacillus detection kit | |
| Bergallo et al. | Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy | |
| KR20140110342A (en) | Primer and probes for the detection of Norovirus, and detection kits and methods thereof | |
| CN110904194A (en) | A combined detection kit for mycoplasma pneumoniae and chlamydia pneumoniae nucleic acid and its application | |
| NL2031088B1 (en) | Locked Nucleic Acid-modified One-step Nested PCR Primers Set and Kit for African Swine Fever Virus | |
| CN114480726B (en) | Primer probe set, kit and detection method for detecting African swine fever virus nucleic acid | |
| CN110951904B (en) | LAMP primer set, kit and detection method for IMP drug-resistant genes resistant to carbapenem antibiotics | |
| CN115786587A (en) | A primer set for simultaneous detection of four pathogens in multiplex PCR, its detection method, and its kit | |
| CN103602737B (en) | A kind of mycobacterium tuberculosis RNA/DNA synchronizes quantitative analysis method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210803 Address after: 430070 No. 1 Lion Rock street, Hongshan District, Hubei, Wuhan Applicant after: HUAZHONG AGRICULTURAL University Applicant after: WUHAN KEQIAN BIOLOGICAL Co.,Ltd. Address before: 430070 No. 1 Lion Rock street, Hongshan District, Hubei, Wuhan Applicant before: HUAZHONG AGRICULTURAL University |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |