CN118581096B - Aptamer and preparation method and application thereof - Google Patents
Aptamer and preparation method and application thereof Download PDFInfo
- Publication number
- CN118581096B CN118581096B CN202411075189.0A CN202411075189A CN118581096B CN 118581096 B CN118581096 B CN 118581096B CN 202411075189 A CN202411075189 A CN 202411075189A CN 118581096 B CN118581096 B CN 118581096B
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- aptamer
- seq
- protein
- marker
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种核酸适配体及其制备方法和应用，涉及核酸适配体制备技术领域，所述核酸适配体包括适配核酸和阻断核酸；所述适配核酸在核心适配核酸的基础上添加修饰段得到，所述核心适配核酸的序列如SEQ ID No.1所示，所述适配核酸和所述阻断核酸通过杂交形成所述核酸适配体，其中，所述适配核酸如SEQ ID No.2所示、所述阻断核酸如SEQ ID No.3所示，该核酸适配体与繁殖型结核分枝杆菌亲和力高，也即与EC蛋白的解离常数高，且灵敏度高、特异性强、成本低廉，适于一线的推广应用。
The invention discloses a nucleic acid aptamer and a preparation method and application thereof, and relates to the technical field of nucleic acid aptamer preparation. The nucleic acid aptamer comprises an aptamer nucleic acid and a blocking nucleic acid; the aptamer nucleic acid is obtained by adding a modified segment on the basis of a core aptamer nucleic acid, the sequence of the core aptamer nucleic acid is shown in SEQ ID No.1, the aptamer nucleic acid and the blocking nucleic acid are hybridized to form the nucleic acid aptamer, wherein the aptamer nucleic acid is shown in SEQ ID No.2, and the blocking nucleic acid is shown in SEQ ID No.3. The nucleic acid aptamer has a high affinity with reproductive Mycobacterium tuberculosis, that is, a high dissociation constant with EC protein, and has high sensitivity, strong specificity, and low cost, and is suitable for first-line promotion and application.
Description
Technical Field
The invention relates to the technical field of preparation of nucleic acid aptamers, in particular to a nucleic acid aptamer, a preparation method and application thereof.
Background
At present, the error or delayed diagnosis of the propagation type mycobacterium tuberculosis is one of the main reasons that the tuberculosis epidemic situation is difficult to quickly decline, so that the accurate identification of the 'life-death' state of the mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is the basis and key for controlling and treating tuberculosis, and has important theoretical and practical significance for quickly detecting infection, guiding clinical medication and curing tuberculosis.
The existing biological diagnosis technology of the mycobacterium tuberculosis is compared and evaluated. Taking the most mainstream Xpert MTB modern molecular diagnostic technology as an example, the technology judges the existence of the mycobacterium tuberculosis by whether the specific genome sequence fragment or the mutation sequence of the drug-resistant gene locus of the mycobacterium tuberculosis can be amplified, so that the propagation type mycobacterium tuberculosis infection and the latent mycobacterium tuberculosis infection cannot be distinguished, namely, the existence of nucleic acid fragments of the mycobacterium tuberculosis can still be detected after the mycobacterium tuberculosis loses the propagation and transmission capacity, and false positives are caused. As another example, traditional isolated culture of Mycobacterium tuberculosis is currently the only identification method that can accurately identify the presence of a propagating Mycobacterium tuberculosis, but requires a longer culture period and is extremely prone to missing the golden time for diagnosis and cure in clinic.
However, active tuberculosis, latent tuberculosis infection and nontuberculous mycobacterial infection all have positive results of mycobacterium tuberculosis in the existing molecular diagnosis, resulting in false positive results of latent tuberculosis infection and nontuberculous mycobacteria. Because the medication scheme, isolation time and treatment cost of the three infection conditions of active tuberculosis, latent tuberculosis infection and nontuberculous mycobacterium infection are completely different, the erroneous or delayed diagnosis result can obviously influence the treatment effect and cause great physical and economic stress to patients, and the problems of high cost, insufficient specificity and sensitivity or expensive matched instruments of the diagnosis technologies seriously obstruct the detection and diagnosis application process in clinical line.
Disclosure of Invention
The invention mainly aims to provide a nucleic acid aptamer, a detection method and application thereof, and aims to solve the problems that in the prior art, positive results of mycobacterium tuberculosis appear in the existing molecular diagnosis, latent infection of the mycobacterium tuberculosis and false positive results of nontuberculous mycobacterium are caused, and the diagnosis technologies have high cost, specificity and sensitivity.
To achieve the above object, the present invention proposes a nucleic acid aptamer for binding to EC protein, the nucleic acid aptamer comprising an aptamer nucleic acid and a blocking nucleic acid;
wherein the nucleic acid aptamer comprises an aptamer nucleic acid and a blocker nucleic acid;
The core matching nucleic acid is obtained by adding a modification section on the basis of the core matching nucleic acid, the sequence of the core matching nucleic acid is shown as SEQ ID No.1, and the matching nucleic acid and the blocking nucleic acid form the nucleic acid aptamer through hybridization, wherein the matching nucleic acid is shown as SEQ ID No.2, and the blocking nucleic acid is shown as SEQ ID No. 3.
In one embodiment, the sequence size of the adapter nucleic acid is 20-30 bp, and/or the sequence size of the blocking nucleic acid is 20-38 bp.
In one embodiment, the sequence of the aptamer comprises a nucleotide sequence labeled with a label comprising a biotin label, a digoxin label, a fluorescent label, a nano luminescent material label, a polyethylene glycol label, a peptide fragment label, a protein label, an enzyme label, or a functional group label, and/or,
Bases in the sequence of the nucleic acid aptamer include phosphorylated bases, oxymethylated bases, methylated bases, aminated bases, sulfhydrylated bases, or isotopically substituted bases.
The invention also provides a preparation method of the nucleic acid aptamer, which comprises the following preparation steps:
s1, providing an adaptive nucleic acid and a blocking nucleic acid, wherein the adaptive nucleic acid is shown as SEQ ID No.2, and the blocking nucleic acid is shown as SEQ ID No. 3;
S2, hybridizing the adapter nucleic acid and the blocking nucleic acid to form a hybridized dimer, so as to obtain the nucleic acid aptamer.
The invention provides a detection method of a nucleic acid aptamer, which is used for identifying a breeding mycobacterium tuberculosis and comprises the following steps:
s10, providing a sample to be tested;
s20, mixing and incubating the sample to be detected with the nucleic acid aptamer to obtain an incubation liquid;
s30, mixing the incubation liquid with isothermal amplification liquid, and performing amplification reaction to obtain an amplification strip;
s40, analyzing and judging the amplification bands to identify whether the breeding type mycobacterium tuberculosis exists or not;
Wherein the nucleic acid aptamer is the nucleic acid aptamer according to any one of the above or the nucleic acid aptamer prepared by the method for preparing the nucleic acid aptamer.
The invention provides application of the aptamer in preparation of a diagnostic reagent, a test strip or a biosensor of mycobacterium tuberculosis.
The invention provides a biological agent for judging tuberculosis typing, wherein the components of the biological agent comprise the nucleic acid aptamer of any one of the above or the nucleic acid aptamer prepared by the preparation method of the nucleic acid aptamer.
The invention discloses a nucleic acid aptamer, which comprises an aptamer nucleic acid and a blocking nucleic acid, wherein the aptamer nucleic acid is obtained by adding a modification section on the basis of a core aptamer nucleic acid, the sequence of the core aptamer nucleic acid is shown as SEQ ID No.1, the aptamer nucleic acid and the blocking nucleic acid form the nucleic acid aptamer through hybridization, the aptamer nucleic acid is shown as SEQ ID No.2, the blocking nucleic acid is shown as SEQ ID No.3, and the obtained nucleic acid aptamer has high affinity with a propagating mycobacterium tuberculosis, namely has high dissociation constant with EC protein, and has high sensitivity, strong specificity and low cost, thereby being suitable for first-line popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flowchart of the Mag-beads SELEX workflow of example 1 provided by the present invention;
FIG. 2 is an electrophoretogram of the nucleic acid aptamer affinity for the EC protein determined by the Mag-beads SELEX system in example 1 provided by the present invention;
FIG. 3 is an electrophoretogram of a tube-type Mycobacterium tuberculosis assay according to example 1 of the present invention by combining isothermal amplification of nucleic acids;
FIG. 4 is an map of an aptamer sequencing alignment in an EC protein binding product in a 15 th round of Mag-beads SELEX screening;
FIG. 5 is a graph showing a curve fit of the binding of SEQ ID No.2 to the EC protein as determined by Isothermal Titration Calorimetry (ITC) in example 1 provided in the present invention;
FIG. 6 shows isothermal amplification bands obtained with different concentrations of EC protein as amplification template;
FIG. 7 is a graph comparing the serum clinical samples of healthy people detected by the invention with the clinical serum samples identified as positive for Mycobacterium tuberculosis by GeneXpert.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
At present, active tuberculosis, latent tuberculosis infection and nontuberculous mycobacterial infection all have positive tuberculosis results in the existing molecular diagnosis, and cause false positive results of the latent tuberculosis infection and nontuberculous mycobacteria. Since the three infection conditions of active tuberculosis, latent tuberculosis infection and nontuberculous mycobacterium infection are completely different in medication scheme, isolation time and treatment cost, erroneous or delayed diagnosis results will significantly affect the treatment effect and cause significant physical and economical stress to patients. In addition, the problems of high cost, insufficient specificity and sensitivity, expensive matched instruments and the like of the diagnosis technologies seriously obstruct the detection and diagnosis application process in the clinical line.
In view of the above, the invention discloses a nucleic acid aptamer, which comprises an aptamer nucleic acid and a blocking nucleic acid, wherein the aptamer nucleic acid is obtained by adding a modification section on the basis of a core aptamer nucleic acid, the sequence of the core aptamer nucleic acid is shown as SEQ ID No.1, and the aptamer nucleic acid and the blocking nucleic acid form the nucleic acid aptamer through hybridization, wherein the aptamer nucleic acid is shown as SEQ ID No.2, and the blocking nucleic acid is shown as SEQ ID No. 3.
It should be noted that, the said adapting nucleic acid is obtained by adding a modification section by using the said core adapting nucleic acid as a template, the sequence of the said core adapting nucleic acid is shown as SEQ ID No.1, the said blocking nucleic acid is complementary with at least part of the said adapting nucleic acid, the said adapting nucleic acid and blocking nucleic acid form a nucleic acid aptamer through hybridization, the aptamer has high affinity with the breeding mycobacterium tuberculosis, namely, has high dissociation constant with EC protein, and has high sensitivity, strong specificity and low cost, and is suitable for popularization and application in first line.
It should be noted that, the aptamer is also called as "nucleic acid antibody", which is a short nucleic acid fragment (the nature of which is DNA or RNA) with a length below 100bp, and after natural folding, a three-dimensional conformation is generated, and a tightly-compatible complex can be formed with a protein, a molecular marker or the surface of a living cell, and the KD value of the affinity dissociation constant can reach picomolar (10 -7 to 10 -9 M) as the highest, and the aptamer is a high-specificity "nucleic acid antibody" of a target molecule, which has high sensitivity and specificity of antigen-antibody reaction, but has lower preparation cost, easier synthesis, preservation and deep design application, however, no report of direct, rapid and sensitive detection of MTB in clinic through the high-specificity aptamer has been found at present.
The dissociation constant of the aptamer obtained by the invention and the EC protein is as high as 43nM (KD (M) =43.0e -9), so that the aptamer can tightly bind (capture) the EC protein secreted by the mycobacterium slipknot in a short time.
Further, the sequence of the core adaptive nucleic acid is shown as SEQ ID No.1, the specific sequence is 5' -GATCGCCAACTTCCATGAGGAGGGGGCTTTCAAGGGCG, any nucleic acid which takes the core adaptive nucleic acid as a template and is subjected to substitution and/or deletion and/or addition of one or more nucleotides is used as first nucleic acid, and the first nucleic acid also has the same or similar functional effect, because the first nucleic acid still maintains the specific binding capacity with the core adaptive nucleic acid after the change, has similar functional effect, and the capability for specifically identifying the breeding type mycobacterium tuberculosis is obviously enhanced. For example, by substituting certain bases with others, the affinity and specificity of the first nucleic acid may be improved, thereby increasing the accuracy and sensitivity of the diagnosis. In addition, experimental data show that the application of the first nucleic acid formed by the change in the aptamer has remarkable advantages, and has important application prospects for detection and identification of the mycobacterium tuberculosis.
The blocking nucleic acid takes the obtained adapter nucleic acid as a template, is at least partially complementary to the adapter nucleic acid, and forms a nucleic acid aptamer through hybridization of the adapter nucleic acid and the blocking nucleic acid.
Specifically, the adaptive nucleic acid is shown as SEQ ID No.2, and the specific sequence is:
5-GATCGCCAACTTCCATGAGGAGGGGGCTTTCAAGGGCGCGACGAAGCTGTTTTATTCTC-3’;
The blocking nucleic acid is shown as SEQ ID No.3, and the specific sequence is as follows:
5’-CTAGCGGTTGAAGGTACTCCTCCCCCGAAAGTTCCCGC-3’;
Annealing the SEQ ID No.2 and the SEQ ID No.3 according to the proportion of 1:1, and then cooling in a gradient way, wherein the hybridization is performed to form a hybridization dimer state of the SEQ ID No.2-SEQ ID No. 3. In some embodiments of the invention, the sequence size of the adapter nucleic acid is 59bp, and can be shortened to core adapter nucleic acid, and the core sequence size of the adapter nucleic acid can be 20bp, 25bp or 30bp.
In some embodiments of the invention, the blocking nucleic acid has a sequence size of 20-38 bp, and in these ranges, the blocking nucleic acid, i.e., the Blocker sequence, works best and an excessively long Blocker sequence is prone to false positives for subsequent nucleic acid amplification.
The sequence of the aptamer comprises a nucleotide sequence marked by a marker, wherein the marker comprises a biotin marker, a digoxin marker, a fluorescent marker, a nano luminescent material marker, a polyethylene glycol marker, a peptide fragment marker, a protein marker, an enzyme marker or a functional group marker, and/or,
Bases in the sequence of the nucleic acid aptamer include phosphorylated bases, oxymethylated bases, methylated bases, aminated bases, sulfhydrylated bases, or isotopically substituted bases.
The modification can be a marker or can phosphorylate or oxymethylate the base, so that the subsequent detection is convenient and the screening is convenient.
The invention also provides a preparation method of the nucleic acid aptamer, which comprises the following preparation steps:
s1, providing an adaptation nucleic acid and a blocking nucleic acid, wherein the adaptation nucleic acid comprises a core adaptation nucleic acid and/or a first nucleic acid, the first nucleic acid takes the core adaptation nucleic acid as a template, any nucleic acid subjected to substitution and/or deletion and/or addition of one or more nucleotides is taken as the first nucleic acid, the sequence of the core adaptation nucleic acid is shown as SEQ ID No.1, and the blocking nucleic acid takes the adaptation nucleic acid as the template and is at least partially complementary with the adaptation nucleic acid;
S2, hybridizing the adapter nucleic acid and the blocking nucleic acid to form a hybridized dimer, so as to obtain the nucleic acid aptamer.
Specifically, the aptamer-blocker is a hybridized dimer of SEQ ID No.2 and SEQ ID No.3, and can be prepared by fully mixing two oligos according to a mol ratio of 1:1, hybridizing for 5 minutes at 95 ℃ and then cooling in a gradient way, wherein the isothermal amplification solution contains recombinase (2 mug/mul), single-chain binding protein (2 mug/mul) and Bsu DNA polymerase (5U/mul), the solution is enzyme-free sterile water, and the concentration ratio of SEQ ID No.2 or SEQ ID No.3 can be increased, reduced or differentially adjusted simultaneously, so that the same or similar functional effect as the invention can be obtained.
The aptamer prepared by the method is an identification scheme which does not depend on nucleic acid extraction and can directly carry out high-specificity propagation type mycobacterium tuberculosis on clinical serum samples. The method does not depend on expensive instruments and equipment, has no wound, greatly shortens the detection operation time, saves the detection cost and widens the application scene.
The invention also provides a detection method of the nucleic acid aptamer, which comprises the following steps:
s10, providing a sample to be tested;
s20, mixing and incubating the sample to be detected with the nucleic acid aptamer to obtain an incubation liquid;
s30, mixing the incubation liquid with isothermal amplification liquid, and performing amplification reaction to obtain an amplification strip;
s40, analyzing and judging the amplification bands to identify whether the breeding type mycobacterium tuberculosis exists or not;
wherein the nucleic acid aptamer is the nucleic acid aptamer according to any one of the above.
The detection method provided by the invention has the advantages that the sensitivity of an aptamer-blocker is high, the detection value of EC protein is 1 ag/mu l (equal to 3 EC protein/mu l, namely 3 EC protein particles/mu l can be detected), the specificity is high, the mycobacterium tuberculosis can be accurately identified from samples such as healthy human serum, nontuberculous mycobacterium infectious human serum, tuberculous mycobacterium positive serum, escherichia coli culture medium filtrate, healthy human cell culture filtrate and the like, whether a patient has active tuberculosis can be accurately judged, and the latent infection of the mycobacterium tuberculosis and the false positive caused by nontuberculous mycobacterium infection can not be caused. The invention has higher reference value for diagnosis and clinical medication of the infection of the reproductive mycobacterium tuberculosis, and is suitable for market popularization and clinical application.
It should be noted that, at present, a scheme for co-screening the mycobacterium tuberculosis DNA nucleic acid aptamer by adding a short nucleic acid blocking strand (blocker) paired with an aptamer sequence has not been reported, because the technical difficulty of introducing the blocker to co-screen the mycobacterium tuberculosis DNA nucleic acid aptamer is to improve the specificity of screening and reduce false positive results caused by non-specific hybridization of the aptamer. The screening result can be judged by detecting the blocking device, compared with the traditional method, the method can improve screening accuracy and reliability, reduce interference sequences, improve screening efficiency, and save time and cost.
It should be noted that, the early secretory antigen target 6kDa protein (ESAT-6) and the culture filtrate protein-10 (CFP-10) are related small secretory proteins found in MTB culture filtrate, the protein size is 22 daltons (kDa), the protein is used as immunodominant antigen, the protein is encoded by Rv3874 and Rv3875 genes at the-1 position of the difference region of the virulent genome of highly pathogenic mycobacterium tuberculosis, the protein exists in a plurality of stages of tuberculosis infection, and the protein is deleted in bacillus calmette-guerin strain and most environmental mycobacterium tuberculosis, and the protein can be used as molecular diagnostic markers to effectively distinguish and identify the mycobacterium tuberculosis. Thus, a 1:1 tightly linked complex (ESAT-6-CFP-10, EC protein) formed after co-transcription of ESAT-6 and CFP-10 with Mycobacterium tuberculosis in the reproductive stage can be used as a marker for identifying whether Mycobacterium tuberculosis is a "living bacterium".
The invention provides application of the aptamer in preparing a mycobacterium tuberculosis diagnostic reagent, a test strip or a biosensor, and the aptamer obtained by the invention can be used for preparing a reagent strip, a diagnostic reagent or a biosensor for the market, so that the aptamer has the same effect as the aptamer and is not repeated herein.
The invention provides a biological agent for judging tuberculosis typing, which comprises the nucleic acid aptamer of any one of the above or prepared by the preparation method of the nucleic acid aptamer, and the biological agent comprises the nucleic acid aptamer, so that the biological agent has the same effect of the nucleic acid aptamer and is not described in detail herein.
In conclusion, the invention designs, screens and prepares a group of aptamer-blocks which are suitable for specifically distinguishing reproductive mycobacterium tuberculosis from non-reproductive mycobacterium tuberculosis based on the EC protein structure specificity of the mycobacterium tuberculosis. Directly adding aptamer-blocker into a clinical serum sample for incubation, then reacting for 30-90 minutes at an ambient temperature of 25-42 ℃ by utilizing an isothermal amplification technology (lsothermal Amplification Technology), and judging whether to carry the breeding mycobacterium tuberculosis according to whether the nucleic acid aptamer fragment is detected
It should be noted that SELEX is a process method for screening nucleic acid aptamer in vitro, and the identification target may be small molecule, polypeptide, protein, cell or bacterial individual. The method mainly comprises ① parts for binding the aptamer to the target molecule, ② parts for eluting unbound aptamer, ③ parts for sequencing and determining the sequence of the aptamer bound to the target molecule. Relates to the synthesis of single-stranded nucleic acid fragment libraries, protein adsorption, polymerase Chain Reaction (PCR) and high-throughput sequencing by chip technology. Wherein, in the ① th aptamer binding to the target molecule portion, in order to fully bind to the target domain, if the aptamer is DNA, the aptamer needs to be treated at high temperature in advance and cooled in gradient to generate a three-dimensional higher conformation with more than two levels.
A group of methods for specifically identifying the aptamer of the mycobacterium tuberculosis comprises the following steps:
(1) Preparing a sample to be detected, wherein the sample to be detected is serum, sputum, urine, bacterial culture solution and the like, wherein the urine and the bacterial culture solution are filtered by a 0.22 mu m filter screen to remove macromolecules in the sample, and the serum and the sputum sample can be immediately subjected to detection operation of mycobacterium tuberculosis only by taking supernatant after centrifugation and are stored at-80 ℃ for a long time;
(2) The aptamer-blocker is incubated, namely 10 mu l of the aptamer-blocker and 200-500 mu l of the sample to be detected in the step (1) are added into a tube, the reaction tube is fully mixed for 30-60 min at 25-42 ℃,20 mu l of the mixed solution is added into 30 mu l of the isothermal amplification solution, and the reaction is immediately continued for 30-60 min at 25-42 ℃;
(3) And judging the result, namely analyzing whether the electrophoresis band is amplified to SEQ ID No.2 by a nucleic acid gel imager, identifying that the sample with positive nucleic acid band exists in the Mycobacterium tuberculosis (positive), and identifying that the sample without positive nucleic acid band is negative in the Mycobacterium tuberculosis.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1 screening of a set of Mycobacterium tuberculosis reproducing nucleic acid aptamers
① Mag-beads SELEX system
Since the EC protein is a specific small molecule protein secreted by mycobacterium tuberculosis in the reproductive stage, the EC protein can be used as a molecular diagnostic marker of "live bacteria". As shown in FIG. 1, the present invention adds a blocker fragment (SEQ ID No. 3) partially complementary to the aptamer sequence (SEQ ID No. 2) to the procedure of screening and purifying the expressed EC protein (purchased from Shanghai Jinuo Biotechnology Co., ltd., product No. 50130E-100) nucleic acid aptamer in the SELEX system in vitro, and forms an aptamer-blocker (SEQ ID No.2-SEQ ID No. 3) hybrid dimer. Thus, the double hybridization product of SEQ ID No.2-SEQ ID No.3 can completely release SEQ ID No.3 after contacting with EC protein, thereby forming EC protein-SEQ ID No.2 polymer, and the fragment of SEQ ID No.3 is detected in the supernatant after separating the protein, and the specific operation steps are as follows:
Randomly generating 59 bp DNA oligo-strand aptamer, designing a plurality of blocks with the length of 21bp based on the aptamer sequence, hybridizing to form a DNA long-short chain dimer library of the aptamer-block, then co-incubating with EC protein, eluting, re-incubating, and eluting Mag-heads SELEX screening, and obtaining the optimal aptamer sequence and the preliminarily matched block sequence thereof after 15 rounds of screening, namely the aptamer nucleic acid of SEQ ID No.2 and the blocking nucleic acid of SEQ ID No. 3.
② Adaptive nucleic acid body screening and identification
As can be seen from the screening of the Mag-beads SELEX system, as shown in FIG. 2, FIG. 2 includes A and B in FIG. 2, wherein A in FIG. 2 is M: DNA LADDER DL:750, and 01 is the amplification product of Aptamer-blocker01 in the first round of EC protein eluent; 02 amplification products of Aptamer-blocker02 in the first round of EC protein eluate; 03 amplification products of Aptamer-blocker03 in the first round of EC protein eluate; 04:the amplification product of Aptamer-blocker04 in the first round of EC protein eluate, 05:the amplification product of Aptamer-blocker05 in the first round of EC protein eluate, 06:the amplification product of Aptamer-blocker06 in the first round of EC protein eluate, 07:the amplification product of Aptamer-blocker07 in the first round of EC protein eluate, 08:the amplification product of Aptamer-blocker08 in the first round of EC protein eluate, 09:the amplification product of Aptamer-blocker09 in the first round of EC protein eluate, 10:the amplification product of Aptamer-blocker10 in the first round of EC protein eluate, A in FIG. 2 is the aptamer and blocker bands in the EC protein eluate after PCR amplification of round 1 Mag-beads EX, the lanes of amplified two distinct bands are the aptamer and blocker bands after the aptamer dimer has been cleaved, and the aptamer band is amplified by one of the aptamer and the aptamer band is amplified by the aptamer and the aptamer band is amplified by the aptamer band is the aptamer band of the first 1 and the aptamer band is the aptamer band of the aptamer band is the band of the aptamer band of the band of band.1 band.1.1.1 band.1 band.band.band.band band amplifier band nucleic amplifier band amplifier nucleic amplifier in an two in an in, 01 amplification product of Aptamer-blocker01 in the second round of EC protein binding product; 02 amplification products of Aptamer-blocker02 in the second round of EC protein binding products; 03:the amplification product of Aptamer-blocker03 in the second round of EC protein glue, 04:the amplification product of Aptamer-blocker04 in the second round of EC protein glue, 05:the amplification product of Aptamer-blocker05 in the second round of EC protein glue, 06:the amplification product of Aptamer-blocker06 in the second round of EC protein glue, 07:the amplification product of Aptamer-blocker07 in the second round of EC protein glue, 08:the amplification product of Aptamer-blocker08 in the second round of EC protein glue, 09:the amplification product of Aptamer-blocker09 in the second round of EC protein glue, 10:the amplification product of Aptamer-blocker10 in the second round of EC protein glue, B in FIG. 2 is the amplification of an Aptamer strip in the second round of EC protein SEL glue after PCR amplification, B in FIG. 2 is the second round of Mag-Beads EC protein strip, and the second strip is the estimated binding of Aptamer-blocker protein in the second round of EC protein glue, 01, lanes are estimated from this amplification of the second round of EC protein strip, 03. The hybridized polymers 07 and 10 released short chain blockers upon affinity with the EC protein, as seen in FIG. 2, two nucleic acid fragments of different sizes were amplified by PCR in the EC protein eluate after the first round of screening as shown in FIG. 2A, the hybridized dimers 03, 07 and 10 of A in FIG. 2 released oligo's blockers from the hybridized dimers into the eluate upon contact with the ESAT6-CFP10 protein, and the No. 03, 07 and 10 were not eluted in the second round of Mag-beads SELEX affinity screening as shown in FIG. 2B, and round 15 of screening by the Mag-beads SELEX system.
FIG. 3 includes FIG. 3A, B, C and D, where FIG. 3A is M DNA LADDER DL750, E1-E15 is the amplification product of Aptamer-blocker01 in the first through fifteenth round of EC protein eluate, and D15 is the amplification product of Aptamer-blocker01 in the tenth round of EC protein gel;
FIG. 3B: M: DNA LADDER DL750,750, E1-E15: amplification products of Aptamer-blocker07 in the first through fifteenth round of EC protein eluate, D15: amplification products of Aptamer-blocker07 in the tenth round of EC protein gel-binding products;
FIG. 3C: M: DNA LADDER DL750,750, E1-E15: amplification products of Aptamer-blocker03 in the first through fifteenth round of EC protein eluate, D15: amplification products of Aptamer-blocker03 in the tenth round of EC protein gel-binding products;
DM DNA LADDER DL in FIG. 3, E1-E15 amplification products of Aptamer-blocker10 in the first through fifteenth round of EC protein eluate, D15 amplification products of Aptamer-blocker10 in the tenth round of EC protein gel;
FIG. 3 shows the Aptamer or blocker bands in the EC protein binding products of the 1 st to 15 th round of Mag-beads SELEX screening, and the 15 th round of Mag-beads SELEX screening after PCR amplification (A in FIG. 3 shows the result of amplifying Aptamer01, C shows the result of amplifying Aptamer 03; B in FIG. 3 shows the result of amplifying Aptamer07, D in FIG. 3 shows the result of amplifying Aptamer 10), as shown in FIG. 3, the Aptamer is 03 and 10 in the precipitate crosslinked with the EC protein, and the Aptamer is not present in the eluate, as shown in right column E15 in FIG. 3 and right column E15 in D in FIG. 3.
FIG. 4 includes, from top to bottom, A, B, C and D of FIG. 4, and is divided into EC-Aptar 01, EC-Aptar 03, EC-Aptar 07 and EC-Aptar 10 sequencing alignment maps of EC protein binding products in 15 th round of Mag-beads SELEX screening, wherein A of FIG. 4 is EC-Aptar 01, and specific sequences thereof are as shown in SEQ ID No. 4:
GACGTTTTGCAAGTGCGGCTACTACAAGCGAAGTTCCATCATTTTAGAGTCATAAACGC。
in FIG. 4, B is EC-Aptamer03, which has the following specific sequences:
GATCGCCAACTTCCATGAGGAGGGGGCTTTCAAGGGCGCGACGAAGCTGTTTTATTCTC, i.e.the adaptor nucleic acid according to the invention SEQ ID No.2.
C in FIG. 4 is EC-Aptamer07, shown in SEQ ID No.5, and its specific sequence is:
CAATTAGACAGGCTCTAAGCTTTGGACCCAGCGTAGAAAGTTTGTTATTTCTTCGGGCT。
d in FIG. 4 is EC-Aptamer10, shown in SEQ ID No.6, and its specific sequence is:
TGTTTACACATATTCTTGCGCATTACTTGCTTTGGCATTATCTGTATCCCCCTCAGCGG。
As shown in FIG. 4, by analysis of the first generation sequencing results, EC-Aptamer03 was amplified in the precipitate crosslinked with the EC protein to be identical to the pro sequence (SEQ ID No. 2), so that EC-Aptamer03 was the optimal affinity Aptamer, that is, SEQ ID No.2 of this example.
③ Determination of aptamer binding force
25. Mu.M of SEQ ID No.2 was titrated for 5. Mu.M of EC protein and the thermodynamic change of both upon contact was determined by Isothermal Titration Calorimetry (ITC). Namely, a certain amount of SEQ ID No.2 is titrated into a reaction tank containing EC protein step by step, and the change of heat released or absorbed in the reaction process is monitored in real time, so that the binding force between the two is measured, and the dissociation constant, the reaction enthalpy change and the reaction entropy change are provided. The results of the titration of SEQ ID No.2 and EC protein are shown in FIG. 5, wherein the abscissa is the molar ratio, the ordinate is the heat change value (unit: kilocalories/mol) of the EC protein titrated by SEQ ID No.2, the graph is a fitting graph of the two binding curves, the relationship between the heat change and the molar ratio is shown, and the binding constant and the thermodynamic parameter are fitted. The dissociation constant (Kd) for binding of SEQ ID No.2 to the EC protein is 43.0e -9 M, the lower the Kd value, the stronger the affinity between molecules. 43.0e -9 M indicates that SEQ ID No.2 has a higher affinity for binding to the EC protein. The enthalpy of binding of SEQ ID No.2 to EC protein is >0, indicating that the binding reaction between the two is endothermic, and that due to gibbs free energy Δg (kcal/mol) <0, SEQ ID No.2 reacts spontaneously with EC protein in the contact environment of the two, and that due to absolute values of-tΔs > Δh, Δg <0 is caused, indicating that entropy changes overcome enthalpy changes, and that the reaction is entropy driven, ionic interactions are the main driving force for the process.
The test results are shown in FIG. 5, wherein SEQ ID No.2 and EC protein have high affinity binding, and dissociation constant 43.0e -9 M indicates that the binding force between the two is strong, and the binding can be effectively carried out at low concentration. 2) The thermodynamic characteristics of the binding reaction are that negative enthalpy change and positive entropy change indicate that the binding reaction is exothermic, and the interaction between molecules tends to be ordered in the binding process along with the increase of system entropy, so that the high-affinity binding between SEQ ID No.2 and EC protein indicates that the binding force of the aptamer is good, and the aptamer has potential application value.
The invention provides an aptamer capable of tightly combining with an EC protein of a molecular diagnostic marker of mycobacterium tuberculosis in a propagation period, wherein the affinity coefficient of an aptamer (SEQ ID No. 2) and the EC protein is up to 43.0e -9 M, and based on the aptamer, the aptamer can be coupled with various downstream molecular detection means to carry out qualitative detection on the mycobacterium tuberculosis in propagation with an EC protein-SEQ ID No.2 complex or SEQ ID No.2 as a target.
Example 2 use of a set of reproducing Mycobacterium tuberculosis nucleic acid aptamers
① Preparation of isothermal amplification reaction system
1) Hybridization dimer, adding 10 mu M of the adapter nucleic acid SEQ ID No.2 and 10 mu M of the blocking nucleic acid SEQ ID No.3 into a hybridization solution (50 mM HEPES (pH 7.4), 500 mM NaCl and 0.05% tween 20), reacting for 5 minutes at 95 ℃, and then cooling to room temperature according to a gradient of-0.1 ℃ per second to complete the hybridization of the double fragments;
2) Sample incubation by gradient dilution of EC protein to 1 ng/. Mu.L, 1 pg/. Mu.L, 1 fg/. Mu.L, 1 ag/. Mu.L with sterile water and mixing with 10. Mu.M of the hybrid dimer of SEQ ID No.2-SEQ ID No.3, respectively, sufficient contact and incubation at room temperature for 30 minutes followed by removal of 20. Mu.L from the incubation as downstream sample to be tested. On the basis, the incubation time of the sample and the hybridization dimer of SEQ ID No.2-SEQ ID No.3 is prolonged/reduced to 20-200 minutes, and the same or similar functional effects as the invention can be obtained;
② EC protein detection system
1) The isothermal amplification reaction components and conditions are EC protein, 10 mu M of hybrid dimer of SEQ ID No.2 and SEQ ID No.3, 2 mu g of recombinase, 2 mu g of single-stranded binding protein, 5UBsu DNA polymerase, and the solution is enzyme-free sterile water, and then all the components are mixed evenly and placed into 38 ℃ rapidly for incubation for 60 minutes. On the basis, the same or similar functional effects as those of the invention can be obtained by adjusting the reaction temperature between 37 ℃ and 42 ℃;
2) The components and conditions of PCR amplification reaction are EC protein, hybridized dimer of SEQ ID No.2 and SEQ ID No.3, 2 XPCR Mix, wherein 2 XPCR Mix contains 50mM KCl, 10mM Tris-HCl, 1.5mM MgCl2, 100. Mu.M dATP, 100. Mu.M dCTP, 100. Mu.M dGTP, 100. Mu.M dTTP, 4UTaq DNA polymerase or purchased in any commercial kit, and then the hybridized dimer and 2 XPCR Mix are placed into a PCR amplification instrument for amplification according to 95 ℃,3 minutes, 95 ℃, 30 seconds, 55 ℃, 30 seconds, 72 ℃,10 seconds (30 cycles), 4 ℃ termination reaction procedure;
example 3 the present invention relates to the feasibility, sensitivity, specificity of detection methods
FIG. 6 includes FIG. 6A, B and C, wherein FIG. 6A is M DNA LADDER DL750,1 is an isothermal amplification band using 1 ng/. Mu.L of EC protein as an amplification template, and 2 is a PCR amplification band using 1 ng/. Mu.L of EC protein as an amplification template;
FIG. 6B: DNA LADDER DL750,750, 1: isothermal amplification strip with 0ng/uL EC protein as amplification template, 2: isothermal amplification strip with 1pg/uL EC protein as amplification template, 3: isothermal amplification strip with 1fg/uL EC protein as amplification template, 4: isothermal amplification strip with 1ag/uL EC protein as amplification template;
In the graph 6, M is DNA LADDER DL, 1 is an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2 and 1 ng/. Mu.L of EC protein, 2 is an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2-SEQ ID No.3 and 1 ng/. Mu.L of EC protein, 3 is an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2 and E.coli culture filtrate grown to an OD value of 0.6-0.8, 4 is an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2-SEQ ID No.3 and E.coli culture filtrate grown to an OD value of 0.6-0.8, 5 is an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2-SEQ ID No.3 and lung cancer human alveolar basal epithelial cell culture filtrate, 6 is an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2-SEQ ID No.3 and lung cancer human alveolar basal epithelial cell culture filtrate, 7 is an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2-SEQ ID No.2 and E.6-6.8, and an isothermal amplification strip after incubation of a single hybrid of SEQ ID No.2-SEQ ID No.2 and a bacterial strain of E.37H of Mycobacterium strain grown to an OD value of 0.6-0.8 is an isothermal amplification strip after incubation of a single hybrid of E.2-SEQ ID No.2 and E.37 to an E.6-0.8.
Feasibility test, 1 ng/. Mu.L EC protein is used as a detection template positive to the breeding mycobacterium tuberculosis, 10 mu M of the aptamer prepared in the embodiment 1 is respectively subjected to isothermal amplification and PCR amplification reaction, whether positive bands are amplified or not is observed through 1.0% or 1.5% agarose gel and an agarose gel imager, and the experimental results are shown in a figure 6, wherein the 1 st (isothermal amplification) and 2 nd (PCR amplification) lanes show nucleic acid bands with the sizes of 59 bp, which indicate that the mycobacterium infected by the sample to be detected is the mycobacterium tuberculosis;
Sensitivity measurement by using enzyme-free sterile water as a detection template negative for Mycobacterium tuberculosis, and using 1pg/μl, 1fg/μl and 1ag/μl of EC protein as a detection template positive for Mycobacterium tuberculosis, isothermal amplification was performed on 10 μM amounts of the aptamer prepared in example 1, and whether positive bands were amplified was observed by 1.0% or 1.5% agarose gel and agarose gel imager.
As shown in the B of FIG. 6, the nucleic acid band with the size of 59 bp does not appear in the lane 1, and the nucleic acid band with the size of 59 bp appears in the lanes 2,3 and 4, which shows that the detection scheme related to the invention can achieve detection and identification of 1 ag/mu L EC protein (namely 3 EC proteins/. Mu.L), and has extremely high sensitivity.
The specificity measurement comprises the steps of taking enzyme-free sterile water as a detection template negative to the reproduction type mycobacterium tuberculosis, taking 1 ng/. Mu.L EC protein as a detection template positive to the reproduction type mycobacterium tuberculosis, separating escherichia coli culture filtrate which grows to OD value=0.6-0.8, separating cell culture filtrate which grows for 48 hours after in vitro passage of lung cancer human alveolar basal epithelial cells, separating H37Rv strain mycobacterium tuberculosis culture filtrate which grows to OD value=0.6-0.8, and carrying out isothermal amplification respectively.
The experimental results are shown as C in FIG. 6, and the nucleic acid bands with the sizes of 59 bp appear in lanes 2 and 8, so that the detection scheme related to the invention can be directly applied to in vitro detection and identification of the Mycobacterium tuberculosis and can only specifically detect the Mycobacterium tuberculosis.
④ Clinical sample validation
Collecting 13 parts of serum of healthy people as a negative reference of the mycobacterium tuberculosis and 13 parts of serum of GeneXpert positive people as a positive reference of the mycobacterium tuberculosis, centrifuging at 3000 rpm for 10 minutes at room temperature, discarding the precipitate, taking 200 mu L of supernatant if macroscopic precipitation appears in the serum, then fully incubating all samples with the SEQ ID No.2-SEQ ID No.3 hybrid dimer at room temperature for 30 minutes sequentially, and then carrying out isothermal amplification to detect positive nucleic acid signals.
The detection results are shown in FIG. 7, wherein M: DNA LADDER DL-750, 1-13, and 1 to 13 parts of the isothermal amplification strips after incubation of the hybrid dimer of SEQ ID No.2-SEQ ID No.3 with serum samples of healthy people, and 14~26:SEQ ID No.2-SEQ ID No.3, and 1 to 13 parts of the isothermal amplification strips after incubation of the hybrid dimer with clinical serum samples identified as positive infection of Mycobacterium tuberculosis by GeneXpert, have significant differences (P < 0.0001), and the detection system of the EC protein of step ② in example 2 is implemented. (specific steps: taking 5-10. Mu.M of the hybrid dimer of SEQ ID No.2 and SEQ ID No.3, mixing and contacting thoroughly at room temperature for 1 hour, then taking 20. Mu.L of the mixture, 2. Mu.g of recombinase, 2. Mu.g of single-stranded binding protein, 5UBsu DNA polymerase, dissolving in sterile water, mixing all the components uniformly and putting into 38 ℃ rapidly, incubating for 60 minutes, and performing nucleic acid running observation result). The detection results are that 13 healthy clinical samples are negative, 13 detected GeneXpert positive samples are 11 positive and 2 negative, and the GeneXpert can only detect the mycobacterium tuberculosis nucleic acid and has interference of mycobacterium tuberculosis 'dead bacteria' nucleic acid fragments, so that the GeneXpert positive cannot completely represent the existence of reproductive type bound mycobacterium in the sample, the result of detecting healthy human serum by the scheme of the invention is the clinical negative of the mycobacterium tuberculosis, and 2 samples (18, 24) without the reproductive type mycobacterium tuberculosis are identified in the GeneXpert positive samples, and the MTB with reproductive capacity is required to be described. If the invention is identified as negative after practice, it is indicated that the sample is free of Mycobacterium tuberculosis. As the non-reproductive MTB nucleic acid residue after death can cause the false positive of GeneXpert, the identification result of the implementation of the invention does not conflict with the detection result of GeneXpert, thus showing that the detection scheme related to the invention can be directly applied to in vitro detection and identification of reproductive mycobacterium tuberculosis, and can only specifically detect the reproductive mycobacterium tuberculosis, thereby providing further clinical analysis and guiding the treatment scheme.
In conclusion, the high sensitivity, high specificity and convenience of the detection of the breeding type mycobacterium tuberculosis under the detection condition based on the SEQ ID No.2-SEQ ID No.3 hybrid dimer are fully shown.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411075189.0A CN118581096B (en) | 2024-08-07 | 2024-08-07 | Aptamer and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411075189.0A CN118581096B (en) | 2024-08-07 | 2024-08-07 | Aptamer and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118581096A CN118581096A (en) | 2024-09-03 |
| CN118581096B true CN118581096B (en) | 2024-11-29 |
Family
ID=92537609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411075189.0A Active CN118581096B (en) | 2024-08-07 | 2024-08-07 | Aptamer and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118581096B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014166558A1 (en) * | 2013-04-09 | 2014-10-16 | Tubitak (Turkiye Bilimsel Ve Teknolojik Arastirma Kurumu) | Dna aptamers to diagnose mycobacterium tuberculosis bacteria and treat tuberculosis disease, specific for m. tuberculosis bacteria |
| CN106047882A (en) * | 2016-06-01 | 2016-10-26 | 湖南大学 | Aptamer group in specific binding with mycobacterium tuberculosis and application of aptamer group |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113699158B (en) * | 2021-09-02 | 2023-08-01 | 中国药科大学 | Nucleic acid aptamer of aristolochic acid A as well as screening method and application thereof |
-
2024
- 2024-08-07 CN CN202411075189.0A patent/CN118581096B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014166558A1 (en) * | 2013-04-09 | 2014-10-16 | Tubitak (Turkiye Bilimsel Ve Teknolojik Arastirma Kurumu) | Dna aptamers to diagnose mycobacterium tuberculosis bacteria and treat tuberculosis disease, specific for m. tuberculosis bacteria |
| CN106047882A (en) * | 2016-06-01 | 2016-10-26 | 湖南大学 | Aptamer group in specific binding with mycobacterium tuberculosis and application of aptamer group |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118581096A (en) | 2024-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102098772B1 (en) | Adenovirus screening method associated gastrointestinal Infections and acute respiratory infections by PNA based real-timc PCR | |
| CN112195521A (en) | DNA/RNA co-database building method based on transposase, kit and application | |
| CN110791578B (en) | CRISPR detection primer set for Bordetella pertussis and use thereof | |
| CN113718045B (en) | DNA fragment, primer, probe and kit for detecting 4 kinds of Bordetella pertussis and specifically detecting Bordetella pertussis and application | |
| CN114381538B (en) | LAMP primer set and detection kit for the detection of Nocardia mallei | |
| CN114134218B (en) | Fluorescent detection method based on CRISPR-Cas12a | |
| CN113881806A (en) | Method and kit for detecting novel coronavirus and 69/70 mutant strain based on CRISPR/Cas12a technology | |
| WO2019001187A1 (en) | Multi-liquid phase gene chip detection primer, kit and method for rapidly distinguishing five pathogens in mouse respiratory tracts | |
| CN114592097B (en) | Primer and probe for identifying novel coronavirus Omicron strain BA.1 and/or BA.3 sublines and application thereof | |
| KR102675157B1 (en) | Method and Kit for Diagnosing Bovine tuberculosis using PNA | |
| CN109402274B (en) | Fluorescent quantitative RT-PCR method for identifying A-type and B-type bovine-derived Pasteurella multocida | |
| CN118166129B (en) | RAA-CRISPR primer combination, reaction system, method and application for identifying mycoplasma gallisepticum wild strain and vaccine nucleic acid | |
| CN114107567A (en) | Virus nucleic acid mutation detection method and application | |
| CN110408629A (en) | Mycobacterium tuberculosis H37Rv coding gene and its application | |
| CN118581096B (en) | Aptamer and preparation method and application thereof | |
| TW202129010A (en) | Method and kit for detection of polynucleotide | |
| CN113817873A (en) | A kind of detection method and kit for new coronavirus D614G mutation | |
| CN118834978B (en) | Kit and method for detecting Streptococcus pyogenes | |
| US20200216881A1 (en) | Compositions and methods for detection of genomic variations | |
| CN114561497B (en) | Primer and probe for identifying novel coronavirus Omicron strain BA.1 subline and application thereof | |
| CN114561496B (en) | Primer and probe for identifying novel coronavirus Delta strain and application of primer and probe | |
| KR102502129B1 (en) | Method and Kit for Identifying the serotype of Actinobacillus pleuropneumoniae causing pneumonia in Pigs using PNA probe | |
| CN111254187A (en) | Multiple PCR detection method for Klebsiella pneumoniae, salmonella and bordetella pneumoniae of rabbit | |
| WO2015046737A1 (en) | Dna aptamer specifically binding to virulence regulatory factor prfa protein of listeria and use thereof | |
| CN107557440B (en) | A loop-mediated isothermal amplification method combining AUDG and self-avoidance molecular recognition system |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |