CN111154836A - Targeted nucleic acid capture and detection methods - Google Patents
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- CN111154836A CN111154836A CN202010108791.5A CN202010108791A CN111154836A CN 111154836 A CN111154836 A CN 111154836A CN 202010108791 A CN202010108791 A CN 202010108791A CN 111154836 A CN111154836 A CN 111154836A
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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]
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- 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
- C12Q1/6869—Methods for sequencing
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Abstract
The invention discloses a targeted nucleic acid capturing and detecting method, wherein the capturing method comprises the following steps: releasing nucleic acids from the biological sample; if the target is RNA, reverse transcription to cDNA is required under the action of reverse transcriptase; carrying out isothermal amplification by using a labeled primer; capturing the target nucleic acid fragment using a capture reagent. The detection method comprises the following steps: releasing nucleic acids from the biological sample; if the target is RNA, reverse transcription to cDNA is required under the action of reverse transcriptase; carrying out isothermal amplification by using a primer which is specifically marked to obtain a target nucleic acid fragment; capturing the target nucleic acid fragment using a capture reagent; the target nucleic acid fragment is detected using a revealing reaction, PCR or sequencing method. The method has strong universality, and can enrich and detect when the sample only contains trace target nucleic acid fragments, and the extraction and detection time of the target nucleic acid fragments is greatly shortened.
Description
Technical Field
The invention relates to the field of medical detection, in particular to a targeted nucleic acid capturing and detecting method.
Background
In nature, whether plants, animals or viruses, nucleic acid plays an irreplaceable role in heredity as the genetic material of most organisms, DNA needs to be extracted from cells in order to research genes of the organisms, the process can be realized by a plurality of methods, and the phenol chloroform extraction method, the centrifugal column method, the magnetic bead method and the like are commonly used at present. The conventional chemical extraction method uses reagents such as phenol, chloroform and the like, has high toxicity, has great influence on the health of personnel after long-time operation, has low recovery rate of nucleic acid and large loss amount, and has poor operation repeatability of different experimenters due to large operation system; the centrifugal column method and the magnetic bead method require more samples, consume more materials, have large nucleic acid loss, are easy to leak when the target nucleic acid amount is low, and cannot obtain target nucleic acid fragments. In addition, when the content of the target nucleic acid is low, the phenomenon that the target nucleic acid exists but cannot be detected can occur due to the limited sensitivity of the detection method, and in order to solve the problems, the application provides a method for capturing and detecting the target nucleic acid.
Disclosure of Invention
The invention aims to provide a method for capturing and detecting target nucleic acid, which solves the problem that the target nucleic acid is difficult to extract and detect when the amount of the target nucleic acid is low.
In order to solve the technical problems, the invention adopts the following technical scheme:
a targeted nucleic acid capture method, comprising:
step a, releasing nucleic acid in a biological sample;
c, adding a labeled primer and isothermal amplification enzyme to amplify a target nucleic acid segment;
step d of capturing the labeled target nucleic acid fragments using a capture reagent. The targeted nucleic acid capture method comprises isothermal amplification, namely nucleic acid amplification, and facilitates subsequent capture of target nucleic acid fragments.
Further, the biological sample in the present application is one selected from the group consisting of cell lines, histological sections, biopsy specimens, formalin-fixed paraffin-embedded tissues, body fluids, feces, urine, plasma, serum, whole blood, isolated blood cells, and cell groups isolated from blood.
Furthermore, if the application targets RNA, a step b of adding reverse transcriptase to reverse transcribe RNA into cDNA is also included between the step a and the step c.
Further, in step a of the present application, the cells are disrupted or lysed by using an enzyme, a surfactant, or an amphiphilic ion method, thereby releasing the nucleic acid in the biological sample. The nucleic acid in the application can be derived from plants, animals, microorganisms or viruses, and a buffer solution can be adopted to treat a biological sample so as to crack cells and release the nucleic acid therein, thereby facilitating subsequent operation.
Further, the capture method of the labeled target nucleic acid fragment in the present application is any one of nucleic acid probe hybridization capture, biotin label capture, digoxigenin label capture, isotope label capture, magnetic bead capture, and antibody capture.
A method of targeted nucleic acid detection comprising:
step a, releasing nucleic acid in a biological sample;
c, carrying out isothermal amplification by using a primer specifically marked to obtain a target nucleic acid fragment;
capturing the labeled target nucleic acid fragments using a capture reagent;
and e, detecting the marked target nucleic acid fragment by using a color development method, a PCR (polymerase chain reaction) method or a sequencing method.
The target nucleic acid capturing method comprises isothermal amplification, namely nucleic acid amplification, so that whether the target nucleic acid fragment exists or not can be conveniently detected subsequently, and the detection limit is reduced.
Furthermore, if the application targets RNA, a step b of adding reverse transcriptase to reverse transcribe RNA into cDNA is also included between the step a and the step c.
Further, the specific marker in the present application is any one of biotin, digoxin, and isotope.
Further, the color development method in the present application is any one of colloidal gold, colloidal silver, silicon quantum dots, germanium quantum dots, cadmium sulfide quantum dots, cadmium selenide quantum dots, cadmium telluride quantum dots, zinc selenide quantum dots, lead sulfide quantum dots, lead selenide quantum dots, indium phosphide quantum dots, and indium arsenide quantum dots.
Compared with the prior art, the invention has the beneficial effects that:
the method has strong universality, can be suitable for nucleic acids of animals, plants and viruses, has strong universality on biological samples, and can be suitable for conventional samples.
The isothermal amplification mode is adopted, and when only a trace amount of target nucleic acid fragments are contained in a sample, enrichment and detection can be realized, and meanwhile, the target nucleic acid fragment capturing and detecting time is obviously shortened.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1:
PCR method for detecting trace target nucleic acid fragment in target sample
Treating Coxsackie group A virus EV71 with the lysate;
adding a marked upstream primer SEQ.ID NO 1: GTGTGTCGTAACGGGCAACTC, a downstream primer SEQ.ID NO 2: TTTCAATTGTCACCATAAGCAGC reverse transcriptase and isothermal amplification enzyme, and treating for 30 minutes;
pulling the target nucleic acid using the capture magnetic beads;
preparing a fluorescent PCR system, adding unmarked upstream and downstream primers, and capturing nucleic acid as a template;
preparing a positive control fluorescent PCR system, adding β -Actin upstream primer SEQ.ID NO 3: GGCGAATTCATGGATGATGATATCGCC and downstream primer SEQ.ID NO 4: CCGCTCGAGCTAGAAGCATTTGCGGTGG, and taking an isothermal amplification product as a template;
preparing a negative control fluorescent PCR system, adding upstream and downstream primers, and adding no template;
amplifying for 35 cycles;
ct of the target gene positive sample is less than 30.
The fluorescence quantitative PCR detection of the method for capturing nucleic acid by adopting the target nucleic acid marker can improve the sensitivity of PCR detection, and the minimum detected virus load can reach 10-100 copies per milliliter of samples.
Example 2:
sequencing method for detecting trace target genes in target sample
Treating Coxsackie group A virus EV71 with the lysate;
adding a labeled upstream primer SEQ.ID NO 1: GTGTGTCGTAACGGGCAACTC, a downstream primer SEQ.ID NO 2: TTTCAATTGTCACCATAAGCAGC transcriptases and isothermal amplification enzymes, and treating for 30 minutes;
pulling off the target nucleic acid using the capture magnetic beads;
drawing nucleic acid, performing terminal completion, and building a library;
sequencing by a sequencer;
sequencing data analysis was performed.
The test technique using the nucleic acid-targeted label-capture method can shorten the sequencing time. The sequencing of the virus gene can be completed only by 4 hours on a nanopore sequencer, which is twice as fast as the current fastest method.
Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure and claims of this application. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.
SEQUENCE LISTING
<110> Sichuan Nor Vast science and technology Limited
<120> method for capturing and detecting target nucleic acid
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<170>PatentIn version 3.5
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tttcaattgt caccataagc agc 23
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ccgctcgagc tagaagcatt tgcggtgg 28
Claims (9)
1. A targeted nucleic acid capture method, comprising:
step a, releasing nucleic acid in a biological sample;
c, adding a labeled primer and isothermal amplification enzyme to amplify a target nucleic acid segment;
step d of capturing the labeled target nucleic acid fragments using a capture reagent.
2. The targeted nucleic acid capturing method of claim 1, wherein the biological sample is one selected from the group consisting of a cell line, a histological section, a biopsy specimen, a formalin-fixed paraffin-embedded tissue, a body fluid, a stool, urine, plasma, serum, whole blood, isolated blood cells, and a group of cells isolated from blood.
3. The method for capturing targeted nucleic acid as claimed in claim 1, wherein step b is further included between step a and step c if RNA is targeted, wherein reverse transcriptase is added to reverse-transcribe RNA into cDNA.
4. The method for capturing targeted nucleic acid according to claim 1, wherein the cells are disrupted or lysed in step a by using an enzyme, a surfactant, or a zwitterion method to release the nucleic acid from the biological sample.
5. The targeted nucleic acid capturing method of claim 1, wherein the labeled target nucleic acid fragment capturing method is any one of nucleic acid probe hybridization capturing, biotin label capturing, digoxigenin label capturing, isotope label capturing, magnetic bead capturing, and antibody capturing.
6. A method of detecting a target nucleic acid, comprising:
step a, releasing nucleic acid in a biological sample;
c, carrying out isothermal amplification by using a primer specifically marked to obtain a target nucleic acid fragment;
capturing the labeled target nucleic acid fragments using a capture reagent;
and e, detecting the marked target nucleic acid fragment by using a color development method, a PCR (polymerase chain reaction) method or a sequencing method.
7. The method of claim 6, further comprising a step b of adding reverse transcriptase to reverse transcribe RNA into cDNA between step a and step c if RNA is targeted.
8. The method for detecting a target nucleic acid according to claim 6, wherein the specific marker is any one of biotin, digoxigenin, and an isotope.
9. The targeted nucleic acid detection method according to claim 6, wherein the color development method is any one of colloidal gold, colloidal silver, silicon quantum dots, germanium quantum dots, cadmium sulfide quantum dots, cadmium selenide quantum dots, cadmium telluride quantum dots, zinc selenide quantum dots, lead sulfide quantum dots, lead selenide quantum dots, indium phosphide quantum dots, and indium arsenide quantum dots.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112266951A (en) * | 2020-11-23 | 2021-01-26 | 广东省农业科学院植物保护研究所 | Extension method of biotin labeled primer and application thereof |
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Application publication date: 20200515 |