CN112226489A - Magnetic bead-based target gene targeted enrichment-based nucleic acid extraction method and application thereof - Google Patents
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Abstract
The invention provides a magnetic bead-based target gene targeted enrichment nucleic acid extraction method and application thereof, in particular application in recombinase isothermal amplification. The nucleic acid extraction method comprises the following steps: the capture probe comprises a promoter and a nucleotide which is complementary with a target gene which are connected in sequence, and the 5' end of the capture probe is modified with amino or carboxyl; connecting the magnetic beads with the capture probes through amino carboxyl condensation reaction to obtain capture magnetic beads; and mixing the capture magnetic beads, the sample lysate and a sample to be detected, heating and washing to obtain the capture magnetic beads combined with the target genes. The obtained capture magnetic beads can be added into isothermal amplification or conventional PCR amplification reaction liquid for amplification detection. The capture probe carrying the promoter remarkably improves the detection sensitivity, and can solve the problem of high-concentration background nucleic acid inhibition of isothermal amplification reaction and the practical application problem of recombinase isothermal amplification by combining with a recombinase isothermal amplification technology.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to a magnetic bead-based target gene enrichment-based nucleic acid extraction method and application thereof.
Background
The PCR amplification technology is an important nucleic acid detection technology, but the previous sample treatment and nucleic acid extraction also play important roles in the nucleic acid detection result. The purity and concentration of nucleic acid extraction directly affect the result of PCR, so that better nucleic acid extraction technology can greatly improve the specificity and sensitivity of PCR detection. In recombinase isothermal amplification, complex background nucleic acid can also cause inhibition of amplification of a target gene, and false negative detection results are brought. Conventional nucleic acid extraction, such as column extraction, extracts total DNA or RNA, which can cause false negative results in samples with high nucleic acid content.
Brittany Rohrman et al found that complex Background DNA inhibited the Amplification of Recombinase isothermal Amplification, thereby rendering the results negative, and often appeared in clinical specimens, affecting the accuracy of clinical specimen detection (see Rohrman, B., & Richards-Kortum, R. (2015.). Inhibition of recombination Polymerase Amplification by Background DNA: A laboratory Flow-Based Method for engineering Target DNA. analytical Chemistry,87(3), 1963-1967.).
The reaction characteristics of the recombinase isothermal amplification make it necessary to accurately control the initial content of nucleic acid in the amplification reaction, which causes great inconvenience for the recombinase isothermal amplification to be used for clinical detection. Meanwhile, recombinase isothermal amplification has certain limitations, for example, CN101333565A discloses a method for isothermal and synchronous amplification detection of nucleic acid and its application. In the method, a primer with a T7 promoter is used for amplifying an RNA sequence, but the method can only amplify the RNA sequence but cannot conveniently amplify DNA isothermally, and a DNA sample can be detected only after being denatured at high temperature.
In addition, the conventional nucleic acid extraction mainly includes a column chromatography and a magnetic bead method, the common extraction method uses whole DNA or whole RNA as an extraction object, and when detection is required for one or more specific genes, such as a specific gene detection diagnostic reagent, the extraction method of whole genome or total RNA is easy to cause loss of the gene to be detected when the extraction method is applied to a trace sample, thereby causing false negative of the PCR result. Thus, several targeted nucleic acid enrichment methods have also emerged. The magnetic bead targeted enrichment technology mostly adopts a biotin-streptavidin-affinity mode to perform targeted gene enrichment, but the biotin-streptavidin-affinity method is not high-temperature resistant and is not suitable for a method for pyrolyzing a sample at high temperature, so that the pyrolysis and nucleic acid extraction of the sample need to be performed step by step, and the operation steps are increased.
Therefore, in order to solve the problem of complex background nucleic acid inhibition recombinase isothermal amplification reaction and improve the sensitivity and accuracy of nucleic acid amplification, the development of a more convenient, rapid and effective nucleic acid targeted enrichment and separation method is urgently needed in the art.
Disclosure of Invention
In view of the problems in the prior art, the capture probe and the magnetic bead are coupled together by the condensation reaction of amino and carboxyl to be mixed with the sample lysate, and the nucleic acid enrichment is completed in one step. Meanwhile, the recombinase isothermal amplification technology is combined for use, so that the problem of amplification inhibition of complex background nucleic acid in a sample can be solved, and the practical application problem of recombinase isothermal amplification is solved. Meanwhile, magnetic bead targeted extraction is combined with transcription and recombinase isothermal amplification, so that a low-abundance sample can be detected, and the application value of recombinase isothermal amplification is improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a magnetic bead-based nucleic acid extraction method for target gene targeted enrichment, which comprises the following steps:
(1) synthesizing a capture probe, wherein the capture probe comprises a promoter and a nucleotide which is complementary to a target gene which are connected in sequence, and an amino group or a carboxyl group is modified at the 5' terminal of the capture probe;
(2) connecting the magnetic beads with the capture probes through amino carboxyl condensation reaction to obtain capture magnetic beads;
(3) mixing the capture magnetic beads, the sample lysate and a sample to be detected, heating, and complementarily pairing the capture probes on the capture magnetic beads and target genes in the sample to be detected;
(4) and removing the sample lysate and impurities to obtain the capture magnetic beads combined with the target genes.
In the present invention, a capture probe is designed according to a nucleic acid sequence to be detected, and a capture sequence carrying a carboxyl group or amino group modification at the 5 'end is synthesized, wherein the 5' end of the capture probe is connected with a promoter, such as a T3 promoter, a T5 promoter, a T7 promoter, an SP6 promoter or a P35 promoter. Coupling a capture probe and magnetic beads with amino or carboxyl through condensation reaction, mixing lysis solution containing the magnetic beads with a sample, heating to enable the capture probe to be combined with a target gene, obtaining the target gene through magnet adsorption, and directly adding the magnetic beads adsorbed with the target nucleic acid into a PCR reaction system.
In the invention, the capture sequence carrying the promoter is adopted, so that the DNA and the RNA can be transcribed, the initial sequence can be amplified and amplified, and the detection sensitivity is improved.
After the capture probe binds to the target gene, when the target gene is DNA, the probe extends out of the target nucleic acid complementary sequence under the action of DNA polymerase, and then the extended capture probe is used as a template to synthesize its complementary strand under the action of single-strand binding protein, recombinase and DNA polymerase. Aiming at a DNA target, capture probes respectively aiming at a sense strand and an antisense strand can be designed at different positions of a sequence, so that targeted gene capture is carried out to the maximum extent; when the target gene is RNA, the probe is extended by reverse transcriptase to obtain a cDNA sequence complementary to the target nucleic acid, and the complementary sequence of the cDNA strand is synthesized by RNase H and DNA polymerase. Then, a large amount of cDNA sequences containing target genes are obtained under the action of RNA polymerase and reverse transcriptase, and the detection sensitivity is greatly improved.
Therefore, the method provided by the invention is used for enriching specific genes, can be applied to recombinase isothermal amplification reaction, avoids amplification inhibition caused by excessive complex background nucleic acid, and can be used for enriching trace target genes in a sample and improving the detection sensitivity. Meanwhile, the target enrichment method can be used as an upstream nucleic acid extraction and purification step for PCR detection, can be combined with nucleic acid amplification methods such as recombinase isothermal amplification and conventional real time PCR, can also be combined with various PCR detection methods, improves the detection effect, and has a wide application range.
In the present invention, when multiplex PCR reaction is required, capture probes for multiple targets can be designed, i.e., different capture probes can be connected to magnetic beads.
Preferably, the promoter in step (1) includes promoter sequences such as T3 promoter, T5 promoter, T7 promoter, SP6 promoter or P35 promoter, etc., but the present invention is not limited to the above-mentioned promoters, and other promoters capable of achieving the same function are also applicable, preferably T7 promoter.
Preferably, the length of the capture probe in step (1) is 20-200 nt, such as 30nt, 50nt, 60nt, 80nt, 100nt, 120nt, 130nt, 150nt, 160nt, or 180 nt.
Preferably, the GC content of the capture probe in step (1) is 30 to 70%, and may be, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or the like.
In a preferred embodiment of the present invention, when the 5' -end of the capture probe is modified with an amino group, the surface of the magnetic bead is modified with a carboxyl group. Correspondingly, when the 5' end of the capture probe is modified with carboxyl, the surface of the magnetic bead is modified with amino.
Preferably, the capture probe is modified at its 5 'end with an amino group, and the free 3' end can be spontaneously extended using the captured nucleic acid sequence as a template, thereby increasing the target gene content.
In addition, the magnetic beads and the capture probes can also be connected with each other by streptavidin, Biotin and Biotin derivatives including Desthio Biotin, Desthio Biotin-TEG, Biotin-TEG and the like, as well as FITC, FITC antibody, FAM antibody and the like.
Preferably, the diameter of the magnetic bead in step (2) is 50 to 1000nm, and may be, for example, 50nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, or the like.
Preferably, the magnetic beads in step (2) may be different types of magnetic beads such as silica-based magnetic beads, polystyrene magnetic microspheres, GMA magnetic beads, or agarose magnetic beads, and the magnetic beads in this embodiment are polystyrene magnetic microspheres.
Preferably, an activating agent is added to activate the carboxyl groups on the magnetic beads or the capture probes before the condensation reaction in step (2).
Preferably, the activator comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and/or N-hydroxysuccinimide (NHS).
In the present invention, the following reaction system can be adopted for the preparation of the capture magnetic beads:
as a preferred technical scheme of the invention, the sample lysate in the step (3) is nuclease-free water containing guanidine isothiocyanate, sodium dodecyl sulfate and tris (hydroxymethyl) aminomethane.
Preferably, the molar concentration of the guanidinium isothiocyanate is 0.1-5M, and may be, for example, 0.1M, 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 4M or 5M.
Preferably, the mass concentration of the sodium lauryl sulfate is 0.1 to 1.5%, and may be, for example, 0.2%, 0.4%, 0.6%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or the like.
Preferably, the molar concentration of tris is 0.05 to 0.15M, and may be, for example, 0.06M, 0.08M, 0.1M, 0.11M, 0.12M, 0.13M, or 0.14M.
Preferably, the pH of the sample lysate in step (3) is 6.0 to 8.0, and may be 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, or 7.9, for example.
Preferably, the heating temperature in step (3) is 80 to 100 ℃, for example, 83 ℃, 85 ℃,87 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 96 ℃, 97 ℃, 98 ℃ or 99 ℃, preferably 95 ℃.
Preferably, the heating time in step (3) is 5-10 min, such as 6min, 7min, 8min or 9 min.
As a preferred technical solution of the present invention, the method for removing the sample lysate and impurities in step (4) comprises: and adsorbing the capture magnetic beads by using a magnet, removing sample lysate and impurities, and adding a cleaning buffer solution for cleaning.
Preferably, the washing buffer comprises Tris-HCl buffer or nuclease-free water or the like.
Preferably, the capture magnetic beads bound with the target gene in the step (4) are stored in a nucleic acid storage solution.
Preferably, the nucleic acid preservation solution comprises nuclease-free water or TE (10mM Tris-HCl with pH 8.0, 1mM EDTA) buffer, and the like.
As a preferred embodiment of the present invention, the nucleic acid extraction method comprises the steps of:
(1) preparing a capture probe, wherein the length of the capture probe is 20-200 nt, the GC content is 30-70%, the capture probe is connected with a T7 promoter, and the 5' end of the capture probe is modified with amino or carboxyl;
(2) modifying amino at the 5' end of the capture probe, and modifying carboxyl on the surface of magnetic beads; or, modifying carboxyl at the 5' end of the capture probe, and modifying amino on the surface of the magnetic bead;
(3) activating carboxyl by using an activating agent, and connecting the magnetic beads with the capture probes through amino carboxyl condensation reaction to obtain capture magnetic beads;
(4) mixing the capture magnetic beads, the sample lysate and a sample to be detected, heating at 80-100 ℃ for 5-10 min, and cooling, wherein the cooling can be natural cooling or program cooling so as to slowly cool the sample; wherein the sample lysate is nuclease-free water containing 0.1-5M guanidine isothiocyanate, 0.1-1.5 mass percent sodium dodecyl sulfate and 0.05-0.15M tris (hydroxymethyl) aminomethane;
(5) adsorbing the magnetic beads by using a magnet, adding a Tris-HCl buffer solution for cleaning, dispersing the captured magnetic beads combined with the target genes into nuclease-free water, suspending the magnetic beads and storing.
In a second aspect, the present invention provides a use of the nucleic acid extraction method according to the first aspect in nucleic acid amplification and/or nucleic acid detection. Wherein the nucleic acid amplification comprises the steps of:
mixing the capture magnetic beads combined with the target genes with a nucleic acid amplification reaction solution to carry out nucleic acid amplification; or separating the target gene from the capture magnetic beads, and then adding a nucleic acid amplification reaction solution for nucleic acid amplification.
In a third aspect, the present invention also provides a kit for amplifying nucleic acid according to the method for extracting nucleic acid of the first aspect, the kit comprising:
the kit comprises nucleic acid targeted enrichment magnetic beads, sample lysate, cleaning buffer solution, nucleic acid preservation solution, amplification reaction solution, amplification primers and amplification buffer solution.
Preferably, the amplification reaction solution includes RNA polymerase, DNA polymerase (strand displacement DNA polymerase), reverse transcriptase, single-strand binding protein, recombinase, RNase H, and exonuclease III.
Preferably, the amplification buffer comprises: tris buffer, dithiothreitol, dNTPs, NTPs, ATP, inositol phosphate and magnesium acetate.
Preferably, the kit further comprises a probe, wherein the probe is a modified fluorescent probe.
In a fourth aspect, the present invention further provides a method for using the kit of the third aspect, specifically comprising the following steps:
mixing the capture magnetic beads combined with the target genes with an amplification buffer solution in the kit; or separating the target gene from the capture magnetic beads, and mixing the target gene with an amplification buffer solution in the kit;
adding RNA polymerase, DNA polymerase (strand displacement DNA polymerase), reverse transcriptase, single-stranded binding protein, recombinase, exonuclease III, amplification primer and fluorescent probe to perform nucleic acid amplification detection on the target gene.
Preferably, the use method further comprises the operation of fluorescence acquisition.
In the invention, 1-10 mu L of magnetic beads can be directly added into a nucleic acid amplification reaction solution after the magnetic beads are captured to capture a target sequence. Alternatively, the target gene is isolated from the capture magnetic beads, for example:
(1) heating to 95 ℃, then quickly cooling to separate a capture sequence from a target sequence, separating the target sequence from a capture magnetic bead, adsorbing the magnetic bead by a magnet, taking 1-10 mu L of supernatant, and adding a nucleic acid amplification reaction solution;
(2) degrading a double-stranded part by using exonuclease III to separate a target sequence from captured magnetic beads, heating to inactivate the exonuclease, taking 1-10 mu L of supernate, and adding a nucleic acid amplification reaction solution;
(3) breaking the nucleic acid sequence by alkali liquor treatment, and adding nucleic acid amplification reaction liquid;
(4) when the target sequence is RNA, DNA is digested by DNase treatment, the RNA is separated from the capture magnetic beads, the DNase is inactivated by heating, 1-10 mu L of supernatant is taken, and the nucleic acid amplification reaction solution is added.
In the present invention, the nucleic acid amplification reaction system may comprise the following components:
preferably, when the detection sequence is DNA, the capture probe is modified at the 5 'end and coupled with magnetic beads, the capture probe contains a promoter sequence, and after the capture probe is combined with the target DNA, the free 3' end takes the target DNA as a template under the action of DNA polymerase including Taq enzyme, BST, BSU, Phi29 and other polymerases to generate a complementary DNA sequence 1.
Then synthesizing DNA sequence 2 complementary to DNA sequence 1 under the action of DNA polymerase, recombinase and single-strand binding protein.
In this case, the DNA sequences 1 and 2 have a promoter sequence, RNA is synthesized by RNA polymerase, and RNA is reverse transcribed into cDNA by reverse transcriptase. The cDNA can be amplified in large quantities by the action of DNA polymerases, recombinases, single-stranded binding proteins.
Preferably, when the detection sequence is RNA, the capture probe is modified at the 5 'end and coupled with magnetic beads, the capture probe contains a promoter sequence, and after the capture probe is combined with the target RNA, the free 3' end generates cDNA by taking the target RNA as a template under the action of a reverse transcriptase reaction system. Then, a DNA sequence complementary to the cDNA strand is synthesized by RNase H and DNA polymerase. In this case, the capture probe sequence and its complementary sequence have a promoter sequence.
Simultaneously, RNA is synthesized under the action of a promoter and RNA polymerase, and the RNA is then reverse transcribed into cDNA. And further entering an isothermal amplification cycle, and carrying out mass amplification on the target fragment under the action of DNA polymerase, recombinase and single-strand binding protein.
In the present invention, the generated DNA sequence is detected in real time using a pair of fluorescent probes.
Preferably, the DNA sequence can be detected by using fluorescent probe carrying apurinic/apyrimidinic modification, and the probe can be cut under the action of exonuclease III or endonuclease IV after being combined with the target DNA sequence.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) in the invention, after a capture sequence containing T7 or other promoters is combined with a target sequence, a sequence complementary with the DNA or RNA target sequence can be generated through the action of DNA polymerase or reverse transcriptase, the sequence complementary with the capture sequence is synthesized again in an isothermal amplification system, so that the promoter sequence is introduced into a sequence to be detected, and the sequence to be detected comprising DNA and RNA is amplified through RNA polymerase, thereby enhancing the detection sensitivity and shortening the detection time.
(2) According to the method for extracting the nucleic acid based on the magnetic bead targeted enrichment target gene, the capture probe is designed according to a nucleic acid sequence to be detected, the capture probe is coupled with the magnetic bead through a condensation reaction and then is mixed with a sample, the target nucleic acid in the sample can be targeted enriched by the method, so that the influence of background nucleic acid on amplification is reduced, the capture probe carrying a promoter is combined with recombinase isothermal amplification, the false negative result of recombinase isothermal amplification detection caused by background nucleic acid complexity can be eliminated, meanwhile, the detection sensitivity can be further improved, and the practicability of the recombinase isothermal amplification is greatly improved.
(3) According to the method for extracting the nucleic acid based on the magnetic bead targeted enrichment target gene, the capture magnetic bead can be directly added into a PCR reaction system, the 3' free sequence of the capture probe can be extended by taking the combined sequence as a template, the initial content of the PCR reaction is amplified, and the detection sensitivity is improved;
(4) the method can target and enrich the target nucleic acid in the sample, is suitable for the targeted enrichment extraction of DNA and RNA, can combine with nucleic acid detection methods such as PCR and isothermal amplification, and is simple, convenient and quick to operate.
Drawings
FIG. 1 is a schematic diagram of the nucleic acid extraction, amplification and detection method of the present invention; wherein, the kit comprises 1-capture probe, 2-magnetic bead, 3-target nucleic acid sequence, 4-RNA sequence, 5-cDNA/RNA composite double strand, 6-cDNA and 7-double strand DNA sequence.
FIG. 2 is a graph showing the fluorescence intensity of a target nucleic acid in a reaction solution after amplification without separating magnetic beads from the target nucleic acid in example 1.
FIG. 3 is a graph showing the fluorescence intensity of target nucleic acid in a reaction solution after separation of magnetic beads from the target nucleic acid and amplification in example 1.
FIG. 4 is a graph showing fluorescence intensity profiles of target nucleic acids in reaction solutions after amplification under different backgrounds in example 2.
FIG. 5 is a graph showing fluorescence intensity obtained after amplification of target RNA in the presence of T7 promoter and in the absence of T7 promoter in example 3.
FIG. 6 is a graph showing fluorescence intensity of amplified products using T3 promoter and T7 promoter, respectively, in example 4.
FIG. 7 is a graph showing fluorescence intensity curves of products obtained after amplification of different mock samples in example 5.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
First, the principle of the nucleic acid extraction, amplification, and detection method according to the present embodiment will be briefly described with reference to fig. 1:
(1) nucleic acid extraction: connecting a capture probe 1 with a promoter on a magnetic bead 2, and forming a capture sequence and the magnetic bead through amino carboxyl condensation reaction; then the capture sequence is complementary and matched with a target nucleic acid sequence 3 in a sample to be detected, so that the enrichment of the target nucleic acid is realized;
(2) amplification: under the action of DNA polymerase or reverse transcriptase, the 3' end of the capture probe begins to extend, then a part of fragments directly begin to amplify under the action of DNA polymerase, recombinase and single-strand binding protein, the other part of fragments are synthesized with sequences complementary to sequences with promoters under the action of strand displacement DNA polymerase, then RNA sequences 4 are obtained by transcription, then cDNA/RNA composite double-strands 5 are obtained under the action of reverse transcriptase, and single-stranded cDNA 6 is obtained under the action of RNase H; then synthesizing a complementary strand under the action of DNA polymerase to obtain a double-stranded DNA sequence 7 and carrying out recombinase amplification;
(3) and (3) detection: detection is performed using a fluorescent probe that is complementary paired to the DNA.
In the following embodiments, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout; all sequences used were synthesized by Shanghai Czeri Bio.
Example 1
In this example, the DNA sequence of the ORF1ab gene of the novel coronavirus was captured in a targeted manner by the nucleic acid extraction method and detected by real time PCR amplification.
1. The DNA sequence of ORF1ab gene was synthesized and ligated into pUC57 plasmid
The DNA sequence of ORF1ab gene is shown below (SEQ ID NO. 1):
TAGATGATGATAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATCAGACAACTACTATTCAAACAATTGTTGAGGTTCAACCTCAATTAGAGATGGAACTTACACCAGTTGTTCAGACTATTGAAGTGAATAGTTTTAGTGGTTATTTAAAACTTACTGACAATGTATACATTAAAAATGCAGACATTGTGGAAGAAGCTAAAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAATGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCCTTAAATAAGGCTACTAACAATGCCATGCAAGTTGAATCTGATGATTACATAGCTACTAATGGACCACTTAAAGTGGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTAAACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAAGGTGAAGACATTCAACTTCTTAAGAGTGCTTATGAAAATTTTAATCAGCACGAAGTTCTACTTGCACC
and (3) primer F: 5'-CTTACTGACAATGTATACA-3' (SEQ ID NO.2)
And (3) primer R: 5'-GTAGCCTTATTTAAGGCTC-3' (SEQ ID NO.3)
A fluorescent probe: 5 '-FAM-AACAGTGGTTGTTAATGCAGCCAAT-BHQ 1-3' (SEQ ID NO.4)
2. Capture probe preparation
The sequence of the capture probe is shown in SEQ ID NO.5 (comprising a T7 promoter, the underlined part is the T7 promoter sequence and the target gene complementary sequence, and the 5' end is connected with an amino group):
5'-NH2C6-TAATACGACTCACTATAGGGAGACAGTGTTTAGCAAGATTGTGTCCGCTT-3', a C linked at the 5' end of the sequence6"C" in (1) refers to a carbon atom other than cytosine; commonly used for modifying amino groups on nucleic acid sequences;
3. preparation of Capture magnetic beads
The preparation system comprises the following steps:
the experimental steps are as follows:
(1) after the reaction system is prepared, uniformly mixing by vortex, incubating for 4h at 37 ℃, and uniformly mixing by vortex for 1 time every 30 min;
(2) after the reaction is finished, centrifuging the magnetic beads on the tube cover and the wall for a short time, placing the tube cover and the wall on a magnetic frame, and discarding the supernatant;
(3) add 500. mu.L of 1M NaHCO3Washing for 2 times at room temperature;
(4) add 500. mu.L of 1M NaHCO3Incubating for 30 min;
(5) adding 500 mu L of nuclease-free water, washing for 1 time at room temperature, and discarding the waste liquid;
(6) adding 500 mu L of 0.01% Proclin resuspended magnetic beads to obtain coupled magnetic beads, and storing at 4 ℃.
4. Sample targeted enrichment
(1) 2mL of plasmid-mimic samples (stored in 0.5M guanidinium isothiocyanate solution) at different concentrations were taken, 106copy/mL, 105copy/mL, 104copy/mL, 103copy/mL, 102copy/mL, add 25. mu.L of magnetic beads with capture probe;
(2) after vortex mixing, incubating for 10min at 92 ℃;
(3) after the reaction is finished, centrifuging the magnetic beads on the tube cover and the wall for a short time, placing the tube cover and the wall on a magnetic frame, and discarding the supernatant;
(4) adding 500 μ L of washing solution (nuclease-free deionized water), separating the magnetic beads on the cover and wall of the collection tube instantaneously, placing on a magnetic rack, and discarding the supernatant;
(5) adding 50 μ L of nucleic acid preservation solution (no nuclease deionized water), shaking, mixing, and storing at 4 deg.C;
magnetic bead and target sequence separation: and taking 25 mu L of nucleic acid magnetic bead mixed solution, and incubating at 95 ℃ for 10 min. Centrifuging, separating magnetic beads and nucleic acid while the solution is hot, and keeping the supernatant.
5. PCR detection
(1) Reagent: taq DNA polymerase, dNTP, MgCl2(Shanghai worker)
(2) The method comprises the following steps:
| component name | Dosage of | Component name | Dosage of | |
| | 1μL | 10 μ M primer F | 1μL | |
| 10xbuffer | 2.5 |
10 μ M primer R | 1μL | |
| MgCl2 | 2μL | 10 mu M fluorescent probe | 0.8μL | |
| dNTP | 1μL | Nucleic acids | 5μL | |
| H2O | 10.7μL |
Adding 5 μ L of nucleic acid separated or not separated from the magnetic beads according to the above system;
(3) and (3) detection procedures:
6. results of the experiment
As shown in FIG. 2, 5. mu.L of a mixture of magnetic beads was added; as shown in FIG. 3, the addition of 5. mu.L of the hot elution supernatant detected the target sequence after a period of amplification, indicating that the capture beads were able to effectively capture and enrich for the target sequence.
Meanwhile, the result also shows that the magnetic beads can be directly added into the PCR reaction solution for PCR detection, and the captured targeting sequence can also be separated from the magnetic beads and then added into a PCR reaction system for detection.
Compared with the two methods, the method has the advantages that the experiment steps can be simplified by directly adding the magnetic beads into the PCR reaction solution, and better amplification sensitivity can be obtained at the same time for the following reasons:
firstly, by a pre-extension step, a sequence complementary with a target gene is extended by taking the 3' end of a capture probe as a primer, so that the initial gene quantity to be detected is increased by one time theoretically, and the detection sensitivity is improved; secondly, the captured target gene is separated from the magnetic beads and then added into the PCR system, and compared with the method of directly adding the magnetic beads into the PCR reaction system, certain gene loss is caused, and the detection sensitivity is reduced.
Example 2
This example was used to compare the effects of isothermal amplification of targeted trapped recombinase with isothermal amplification of column-bound extracted recombinase. The hela cell DNA is used as background DNA, and the influence on the detection of the isothermal amplification of the recombinant enzyme is simulated under the condition of a complex background DNA sample.
The plasmid of example 1 was used asAnd detecting the target gene. Sample 1 to be tested contained hela cells at 1X 10910000 copies/mL of plasmid; sample 2 to be tested contained hela cells at 3X 10810000 copies/mL of plasmid; the sample 3 to be tested contains hela cells 109(iv)/mL and plasmid 1000 copies/mL; the sample 4 to be tested contains hela cells 3X 108(iv)/mL and plasmid 1000 copies/mL; wherein the plasmid is the plasmid containing the ORF1ab gene sequence in example 1.
And (3) carrying out sample treatment by adopting a conventional nucleic acid column extraction method and a targeted enrichment method and carrying out recombinase isothermal amplification experiments. The experimental steps are as follows:
1. preparing targeted capture magnetic beads according to the method described in example 1; the capture probe sequence was identical to the capture sequence in example 1 (SEQ ID NO. 5).
2. Sample extraction
a) A targeted enrichment method: taking 2mL of each concentration sample, and carrying out targeted enrichment according to the example 1;
b) column extraction method: taking 2mL samples with various concentrations, and extracting nucleic acid by using a purchased commercial DNA extraction kit (a DNA column extraction kit is purchased from Tiangen Biotechnology (Beijing) Co., Ltd.) according to an operation instruction; in both extraction methods, 50. mu.L of nucleic acid preservation solution was added and stored at 4 ℃.
3. Recombinase isothermal amplification detection
(1) An amplification system:
60mM Tris (pH 7.2), 6mM dithiothreitol, 5% polyethylene glycol, 5mM ATP, 1.5mM dNTPs, 100. mu.M phosphoinositide, 50 ng/. mu.L single-stranded binding protein SSB, 10 ng/. mu.L RecQ, 50 ng/. mu.L UvsX, 50 ng/. mu.L UvsY, 70 ng/. mu.L BSU, 10U RNase H, 1U exonuclease III, 1. mu.M primer F, 1. mu.M primer R, 0.8. mu.M fluorescent probe, 20mM magnesium acetate;
after preparation, 35. mu.L of the sample extract was added thereto, and 5. mu.L of the sample extract was added.
And (3) primer F: 5'-GTTGTTCAGACTATTGAAGTGAATAGTTTTA-3' (SEQ ID NO.6)
And (3) primer R: 5'-GTAGCCTTATTTAAGGCTCCTGCAACACCTCCTC-3' (SEQ ID NO. 7);
a fluorescent probe:
5′-GTGGAAGAAGCTAAAAAGGTAAAACCAACAG/i6FAMdT/G/idSp/iBHQ1dT/TGTTAATGCAGCCAAC3 Spacer-3′(SEQ ID NO.8)
wherein: i6FAMdT is thymine T-residue modified by 6-FAM fluorescein; idSp is an alkali-free tetrahydrofuran THF residue; iBHQ1dT is a thymine T-residue modified by BHQ1 quenching group; c3Spacer represents C3"C" in (1) refers to a carbon atom other than cytosine.
And (3) detection procedures:
| step (ii) of | Procedure | Temperature of | Time of day | Number of |
| 1 | Preheating | 40 |
1 second | 1 |
| 2 | Extension and fluorescence acquisition | 40℃ | 30 |
30 |
4. Results of the experiment
As shown in fig. 4, the curves "magnetic bead-1", "magnetic bead-2", "magnetic bead-3" and "magnetic bead-4" respectively represent the experimental results of the samples to be tested 1 to 4 extracted by the magnetic beads, and the curves "column-1", "column-2", "column-3" and "column-4" represent the experimental results of the samples to be tested 1 to 4 extracted by the column method;
in the figure, when a sample of high-concentration DNA is processed, a column extraction method is adopted for nucleic acid extraction, complex background DNA contained in an extracting solution obviously inhibits the amplification efficiency of recombinase isothermal amplification, and nucleic acid targeted enrichment is adopted, so that the background DNA can be removed, only target genes are reserved, recombinase isothermal amplification can be well amplified, and the detection advantages of the recombinase isothermal amplification can be fully exerted.
Example 3
The nucleic acid extraction method described in this example was used to target capture the RNA sequence of the N gene of a novel coronavirus. The effect of using capture probes containing promoter sequences versus no promoter sequences on amplification sensitivity was also compared.
1. The N gene DNA sequence was synthesized, and the promoter sequence T7 (underlined sequence: T7 promoter) was added to the 5' end of the sequence, which was ligated into the pUC57 plasmid
The DNA sequence of the N gene is shown as follows (SEQ ID NO. 9):
TAATACGACTCACTATAGGGTAATACGACTCACTATAGGGGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGC
and (3) primer F: 5'-TCATCACGTAGTCGCAACAGTTCAAGAAATTCA-3' (SEQ ID NO. 10);
and (3) primer R: 5'-GCCTTTACCAGACATTTTGCTCTCAAGCTGGTTC-3' (SEQ ID NO. 11);
a fluorescent probe:
5′-GCAGCAGTAGGGGAACTTCTCCTGCTAGAA/i6FAMdT/G/idSp/C/iBHQ1dT/GGCAATGGCGGTGAT C3 Spacer-3′(SEQ ID NO.12)；
2. n Gene RNA sequence preparation
RNA Transcription was performed using the TranscriptAId T7 High Yield Transcription Kit (Thermo Scientific) according to the procedure of the Kit, and diluted with nuclease-free water to 1000 copies/mL, 300 copies/mL and 100 copies/mL, respectively.
3. Preparation of capture magnetic beads:
the preparation method of the capture magnetic beads is the same as that described in example 1;
sequence of capture Probe comprising T7 promoter sequence (SEQ ID NO.13)5' -NH2C6-TAATACGACTCACTAT AGGGCTCAGCAGCAGATTTCTTAGTGACAGTTTGGC-3′
Sequence of the capture probe without the T7 promoter sequence (SEQ ID NO. 14): 5' -NH2C6-CTCAGCAGCAGATTTCTTAGTGACAGTTTGGC-3′
4. Targeted capture of RNA sequences
Taking 2mL of prepared RNA products with different concentrations, respectively adding 25 mu L of magnetic beads with capture sequences, uniformly mixing by vortex, and incubating for 10min at 92 ℃;
after the reaction is finished, centrifugally collecting magnetic beads on a tube cover and a wall, placing the tube cover and the wall on a magnetic frame, and discarding supernatant;
adding 500 μ L of lotion, centrifuging to collect magnetic beads on the tube cover and wall, placing on a magnetic rack, discarding the supernatant, and repeating once; adding 50 μ L of nucleic acid preservation solution, shaking, mixing, and storing at 4 deg.C;
5. PCR detection
The PCR reaction system comprises: 60mM Tris (pH 7.2), 6mM dithiothreitol, 5% polyethylene glycol, 5mM ATP, 1.5mM dNTPs, 0.8mM NTPs, 100. mu.M phosphoinositide, 50 ng/. mu.L single-stranded binding protein SSB, 10 ng/. mu.L RecQ, 50 ng/. mu.L UvsX, 50 ng/. mu.L UvsY, 70 ng/. mu.L BSU, 50U T7 RNA Polymerase, 10U M-MLV, 10U RNase H, 1U exonuclease III, 1. mu.M primer F, 1. mu.M primer R, 0.8. mu.M fluorescent probe, 20mM magnesium acetate.
After preparation, 35. mu.L of the sample extract was added thereto.
The reaction procedure is as follows:
| step (ii) of | Procedure | Temperature of | Time of day | Number of |
| 1 | Preheating | 40 |
1 second | 1 |
| 2 | Extension and fluorescence acquisition | 40℃ | 30 |
30 |
6. Results of the experiment
The results of the experiment are shown in FIG. 5: group 1 indicates the capture sequence with the T7 promoter, group 2 indicates the capture sequence without the T7 promoter;
by comparing the capture sequences with the T7 promoter and the T7-free promoter, the sensitivity of the sequence with the T7 promoter to the detection of the target RNA is higher, and the target sequence can be enlarged under the action of the promoter and RNA transcriptase, so that the detection efficiency is effectively improved.
Example 4
The nucleic acid extraction method described in this example was used to target capture the DNA sequence of the new coronavirus N gene. The T3 promoter is adopted to compare the effect with that of the T7 promoter, and a targeting probe with a disordered sequence is adopted as a negative control to carry out a targeting capture specificity test.
1. The PUC57 plasmid containing the sequence of the new coronavirus N gene of example 3 was used, and the new coronavirus N gene was used as a detection target gene. Human genome (Hela cell, 10)9cells/ml), and 107The CFU/mL bacteria include Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC 6538P), Salmonella enteritidis (GEM1.345), Clostridium difficile (ATCC9689), Clostridium perfringens (ATCC13124), 0.2mL each are mixed as background gene, and 0.8mL of 1 × 10 bacteria are added6copy/mL, 1X 105Two mock samples containing different concentrations of N gene were prepared by copying/mL PUC57 plasmid.
2. Preparing targeted capture magnetic beads according to the method described in example 1; the target capture probe sequence using the T7 promoter was the same as the target capture sequence in example 3, with the T7 promoter capture sequence of SEQ ID No.13 and the promoterless capture sequence of SEQ ID No. 14;
the targeting sequence using the T3 promoter was as follows (the underlined part is the T3 promoter sequence):
5'-NH2C6-AATTAACCCTCACTAAAGGGCTCAGCAGCAGATTTCTTAGTGACAGTTTGGC-3'(SEQ ID NO.15), a C linked at the 5' end of the sequence6"C" in (1) refers to a carbon atom other than cytosine.
Meanwhile, a capture sequence of a scrambled sequence was used as a negative control (containing a T7 promoter), and the sequence was:
5'-NH2C6-TAATACGACTCACTATAGGGCGATTGCCATAGGTTCTACGATTGGCATGCAT-3′(SEQ ID NO.16)
3. sample extraction: taking 2ml samples with various concentrations, and carrying out targeted enrichment according to the embodiment 1; finally, 50. mu.L of nucleic acid preservation solution was added and stored at 4 ℃.
4. Recombinase isothermal amplification detection
(1) An amplification system:
60mM Tris buffer pH 7.2, 6mM dithiothreitol, 5% polyethylene glycol, 5mM ATP, 1.5mM dNTPs, 0.8mM NTPs, 100. mu.M phosphoinositide, 50 ng/. mu.L single stranded binding protein SSB, 10 ng/. mu.L RecQ, 50 ng/. mu.L UvsX, 50 ng/. mu.L UvsY, 70 ng/. mu.L BSU, 10U M-MLV, 10U RNase H, 1U exonuclease III, 1. mu.M primer F, 1. mu.M primer R, 0.8. mu.M fluorescent probe, 20mM magnesium acetate, T7 promoter targeted capture sequence group Add 50U T7 RNA Polymerase/T3 promoter targeted capture sequence group Add 50U T3 RNA Polymerase.
After preparation, 35. mu.L of the sample extract was added thereto, and 5. mu.L of the sample extract was added.
(2) Primers and probes
And (3) primer F: SEQ ID No.10, primer R: SEQ ID NO.11, fluorescent Probe: SEQ ID No. 12; and (3) detection procedures:
| step (ii) of | Procedure | Temperature of | Time of day | Number of |
| 1 | Preheating | 40 |
1 second | 1 |
| 2 | Extension and fluorescence acquisition | 40℃ | 30 |
30 |
5. Results of the experiment
As shown in FIG. 6, it was found by comparison that the captured sequence using the T3 promoter and the captured sequence using the T7 promoter have similar amplification effects, and different promoters can cooperate with corresponding RNA polymerase to have relatively similar effects, and can be selected according to practical applications. Meanwhile, the control sequence carrying the T7 promoter has no amplification signal, which indicates that the method has good amplification specificity.
Example 5
In this example, a target capture sequence carrying a promoter sequence was used to detect bacterial target genes in fecal samples. And (3) verifying whether the magnetic bead targeted capture recombinase isothermal amplification method can be applied to complex sample detection or not by detecting and comparing the feces simulation sample and the physiological saline simulation sample. In this example, the tcdB gene of Clostridium difficile (ATCC9689) was examined.
1. Taking semen glycines sample as feces sample of health personnel, weighing, adding 500 μ L of 5 × 10 semen glycines sample respectively4CFU/mL、500μL 5x103CFU/mL Clostridium difficile, and 500. mu.L of 5X10 was added to the same amount of physiological saline4 CFU/mL、500μL 5×103C, CFU/mL clostridium difficile, and a group without clostridium difficile is used as a negative control.
The tcdB gene sequence of the clostridium difficile is SEQ ID NO.17, in particular to
ATAAAATTAGTTATGAAGCAGCATGTAACTTATTTGCAAAGACTCCTTATGATAGTGTACTGTTTCAGAAAAATATAGAAGATTCAGAAATTGCATATTATTATAATCCTGGAGATGGTGAAATACAAGAAATAGACAAGTATAAAATTCCAAGTATAATTTCTGATAGACCTAAGATTAAATTAACATTTATTGGTCATGGTAAAGATGAATTTAATACTGATATATTTGCAGGTCTTGATGTAGATTCATTATCCACAGAAATAGAAACAGCAATAGATTTAGCTAAAGAGGATATTTCTCCTAAGTCAATAGAAATAAATTTATTAGGATGTAATATGTTTAGCTACTCTATCAACGTAGAGGAGACTTATCCTGGA
And (3) primer F: GATTAAATTAACATTTATTGGTCATGGTAAAGA (SEQ ID NO.18)
And (3) primer R: TCCTCTACGTTGATAGAGTAGCTAAACATATTA (SEQ ID NO.19)
Fluorescent probe SEQ ID NO. 20:
TTAATACTGATATATTTGCAGGTCTTGATG/i6FAMdT/A/idSp/A/iBHQ1dT/TCATTATCCACAGAA C3 Spacer
2. preparation of capture magnetic beads:
the preparation method of the capture magnetic beads is the same as that described in example 1;
sequence of the capture probe with the T7 promoter sequence:
(5'NH2C6)TAATACGACTCACTATAGGGTATAATCCTGGAGATGGTGAAATACAAGAAATAGACAAGT(SEQ ID NO.21)
3. tcdB gene capture of stool simulation sample and normal saline simulation sample
Sample treatment: adding 100 mu L of 0.5mM glass spheres to the sample, and adding 1mL of a lysis solution, wherein the lysis solution is 4mol/L guanidinium isothiocyanate, 0.1% SDS and 20mM Tris-HCl; shaking and mixing in an oscillator for 10 min; placing in a high-speed centrifuge at 12000rpm, and centrifuging for 5 min; the supernatant was removed and placed in a new 1.5mL EP tube.
Targeted capture purification: adding 25 mu L of magnetic beads with capture sequences into the sample, uniformly mixing by vortex, and incubating for 10min at 95 ℃;
after the reaction is finished, centrifugally collecting magnetic beads on a tube cover and a wall, placing the tube cover and the wall on a magnetic frame, and discarding supernatant; adding 500 μ L of lotion, centrifuging to collect magnetic beads on the tube cover and wall, placing on a magnetic rack, discarding the supernatant, and repeating once; adding 50 μ L of nucleic acid preservation solution, shaking, mixing, and storing at 4 deg.C;
4. recombinase isothermal amplification detection
(1) An amplification system:
60mM Tris buffer pH 7.2, 6mM dithiothreitol, 5% polyethylene glycol, 5mM ATP, 1.5mM dNTPs, 0.8mM NTPs, 100. mu.M phosphoinositide, 50 ng/. mu.L single-stranded binding protein SSB, 10 ng/. mu.L RecQ, 50 ng/. mu.L UvsX, 50 ng/. mu.L UvsY, 70 ng/. mu.L BSU, 50U T7 RNA Polymerase, 10U M-MLV, 10U RNase H, 1U exonuclease III, 1. mu.M primer F, 1. mu.M primer R, 0.8. mu.M fluorescent probe, 20mM magnesium acetate.
After preparation, 35. mu.L of the sample extract was added thereto, and 5. mu.L of the sample extract was added.
And (3) detection procedures:
| step (ii) of | Procedure | Temperature of | Time of day | Number of |
| 1 | Preheating | 40 |
1 second | 1 |
| 2 | Extension and fluorescence acquisition | 40℃ | 30 |
30 |
5. The experimental results are as follows: as shown in fig. 7, 1# is the result of the stool simulation sample, and 2# is the result of the saline simulation sample. The detection results of the fecal sample and the physiological saline sample are shown through comparison, the target capture recombinase isothermal amplification by the magnetic beads can effectively enrich target sequences in the fecal sample and the physiological saline sample, and can remove the influence of complex components in the sample on detection. The method can be used for nucleic acid detection of pathogenic microorganisms in clinical samples.
In conclusion, the magnetic bead-based nucleic acid extraction method for target gene targeted enrichment can effectively enrich target nucleic acids, amplify target nucleic acid sequences including DNA and RNA, achieve amplification, eliminate recombinase isothermal amplification detection false negative results caused by overhigh background nucleic acids, and improve detection sensitivity.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. A nucleic acid extraction method based on magnetic bead targeted enrichment of target genes is characterized by comprising the following steps:
(1) synthesizing a capture probe, wherein the capture probe comprises a promoter sequence and a nucleotide which is complementary to a target gene which are connected in sequence, and an amino group or a carboxyl group is modified at the 5' terminal of the capture probe;
(2) connecting magnetic beads modified by carboxyl or amino with the capture probes through amino-carboxyl condensation reaction to obtain capture magnetic beads;
(3) mixing the capture magnetic beads, the sample lysate and a sample to be detected, heating, and complementarily pairing the capture probes on the capture magnetic beads and target genes in the sample to be detected;
(4) and removing the sample lysate and impurities to obtain the capture magnetic beads combined with the target genes.
2. The method for extracting nucleic acid according to claim 1, wherein the promoter in step (1) comprises a T3 promoter, a T5 promoter, a T7 promoter, an SP6 promoter or a P35 promoter, preferably a T7 promoter;
preferably, the length of the capture probe in the step (1) is 20-200 nt;
preferably, the GC content of the capture probe in the step (1) is 30-70%.
3. The method of claim 1 or 2, wherein in step (1), the 5' -end of the capture probe is modified with a carboxyl group, and the surface of the magnetic bead is modified with an amino group;
preferably, the 5' -end of the capture probe in step (1) is modified with an amino group, and the surface of the magnetic bead is modified with a carboxyl group;
preferably, the diameter of the magnetic beads in the step (2) is 50-1000 nm;
preferably, the magnetic beads in step (2) comprise any one or a combination of at least two of silica-based magnetic beads, polystyrene magnetic microspheres, GMA magnetic beads or agarose magnetic beads;
preferably, before the condensation reaction in step (2), an activating agent is added to activate the carboxyl on the magnetic beads or the carboxyl on the capture probes;
preferably, the activator comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or N-hydroxysuccinimide.
4. The method for extracting nucleic acid according to any one of claims 1 to 3, wherein the pH of the sample lysate obtained in step (3) is 6.0 to 8.0;
preferably, the sample lysate of step (3) is nuclease-free water containing guanidine isothiocyanate, sodium dodecyl sulfate and tris (hydroxymethyl) aminomethane;
preferably, the molar concentration of the guanidinium isothiocyanate is 0.1-5M;
preferably, the mass concentration of the sodium dodecyl sulfate is 0.1-1.5%;
preferably, the molar concentration of the tris is 0.05-0.15M;
preferably, the heating temperature in the step (3) is 80-100 ℃, and preferably 95 ℃;
preferably, the heating time in the step (3) is 5-10 min.
5. The method for extracting nucleic acid according to any one of claims 1 to 4, wherein the method for removing the sample lysate and impurities in step (4) comprises:
adsorbing the capture magnetic beads by using a magnet, removing sample lysate and impurities, and adding a cleaning buffer solution for cleaning;
preferably, the capture magnetic beads bound with the target gene in the step (4) are stored in a nucleic acid storage solution.
6. The method for extracting nucleic acid according to any one of claims 1 to 5, comprising the steps of:
(1) synthesizing a capture probe, wherein the length of the capture probe is 20-200 nt, the GC content is 30-70%, and the capture probe is connected with a T7 promoter;
(2) modifying amino at the 5' end of the capture probe, and modifying carboxyl on the surface of magnetic beads; or, modifying carboxyl at the 5' end of the capture probe, and modifying amino on the surface of the magnetic bead;
(3) activating carboxyl by using an activating agent, and connecting the magnetic beads with the capture probes through amino carboxyl condensation reaction to obtain capture magnetic beads;
(4) mixing the capture magnetic beads, the sample lysate and a sample to be detected, heating at 80-100 ℃ for 5-10 min, and cooling;
wherein the sample lysate is nuclease-free water containing 0.1-5M guanidine isothiocyanate, 0.1-1.5 mass percent sodium dodecyl sulfate and 0.05-0.15M tris (hydroxymethyl) aminomethane;
(5) adsorbing the magnetic beads by using a magnet, adding a Tris-HCl buffer solution for cleaning, dispersing the captured magnetic beads combined with the target genes into nuclease-free water, suspending the magnetic beads and storing.
7. Use of the nucleic acid extraction method of any one of claims 1 to 6 for nucleic acid amplification and/or nucleic acid detection.
8. A kit for amplifying a nucleic acid by the method for extracting a nucleic acid according to any one of claims 1 to 6, comprising:
the kit comprises nucleic acid targeted enrichment magnetic beads, sample lysate, cleaning buffer solution, nucleic acid preservation solution, amplification reaction solution, amplification primers and amplification buffer solution.
9. The kit according to claim 8, wherein the amplification reaction solution comprises RNA polymerase, DNA polymerase, reverse transcriptase, single-strand binding protein, recombinase, RNase H and exonuclease III;
preferably, the amplification buffer comprises: tris buffer, dithiothreitol, dNTPs, NTPs, ATP, inositol phosphate and magnesium acetate;
preferably, the kit further comprises a probe, wherein the probe is a modified fluorescent probe.
10. A method of using the kit of claim 8 or 9, comprising the steps of:
mixing the capture magnetic beads combined with the target genes with an amplification buffer solution in the kit; or separating the target gene from the capture magnetic beads, and mixing the target gene with an amplification buffer solution in the kit;
adding an amplification reaction solution, an amplification primer and a fluorescent probe to perform nucleic acid amplification on the target gene;
preferably, the use method further comprises the operation of fluorescence acquisition.
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