CN117867663A - Method for constructing specificity library based on Tn7-like transposon and application - Google Patents
Method for constructing specificity library based on Tn7-like transposon and application Download PDFInfo
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Abstract
The invention discloses a method for constructing a specific library based on Tn7-like transposons, which comprises the following steps: (1) Aiming at pathogens to be detected, in a genome conservation area, aiming at more than one conservation sequence, designing a plurality of specific target DNAs (deoxyribonucleic acids) with the PAM sequences of NGTN and a plurality of sgRNAs, wherein the target DNAs are alternately arranged according to forward target DNAs and reverse target DNAs; (2) Constructing a recombinant vector by using the inserted fragment LE-adaptor A-adaptor B-RE; (3) The recombinant vector performs transposition reaction with a nucleic acid sample in a CRISPR-Cas system, and inserts an insertion fragment into a conserved sequence; the CRISPR-Cas system comprises a purified Cas12K protein, a tnsB, tnsC, tniQ protein, a plurality of sgRNAs, a recombinant vector and a nucleic acid sample; (4) And (3) performing PCR amplification to obtain DNA fragments with two ends respectively being the connector A and the connector B, and purifying an amplified product to obtain a specific library. The specific library can be used for detecting pathogens, can detect a plurality of targets, has high detection sensitivity, eliminates the false positive effect caused by non-specific amplification of PCR, and has good application prospect.
Description
Technical Field
The invention relates to a library construction method based on Tn7-like transposon and specific gRNA insertion connector target sequence and then utilizing PCR amplification and application thereof in mycobacterium tuberculosis detection.
Background
High throughput sequencing technology, also known as next generation sequencing technology, has radically changed genomics, transcriptomics and multiunit research, and has wide application in many biomedical research fields. In high throughput sequencing, library construction is a crucial step, and typically library construction employs a linker comprising a specific base sequence attached to both ends of genomic fragment DNA, and then specific primer rows are designed for PCR amplification of the linker sequences to obtain a library for high throughput sequencing. The traditional library building method has the problems of low efficiency, complicated steps and the like.
The transposon database construction technology is to randomly insert transposons in a complex genome by utilizing the insertion capacity of the transposons, thereby generating a large number of mutants and providing a technical means for efficiently and rapidly carrying out functional gene identification on the whole genome for researchers. The Tn7-like transposon (Tn 7-like) transposition which is jointly involved in regulation and control of the proteins coded by the genes containing the genes tnsB, tnsC and tniQ can accurately and efficiently insert exogenous DNA fragments, and the specific insertion of the auxiliary Tn7-like transposon comprises specific linker sequence fragments by combining with the specific binding function of Cas12K, and then a library is obtained by a PCR amplification mode. The method can insert the linker sequence with high efficiency.
The detection of pathogenic nucleic acids usually employs a specific primer amplification method, but conventional amplification methods can only be aimed at a single specific target, and there may be cases of missed detection of variant strains.
Disclosure of Invention
The invention aims to provide a high-throughput sequencing method for constructing a specific library based on a Tn7-like transposon and application thereof, and provides a library construction method for obtaining a specific pathogen sequence by PCR amplification by inserting the Tn7-like transposon into a marker genome for the first time.
The invention designs a plurality of specific gRNAs aiming at a pathogen genome conserved region (within 150 bp), inserts a fragment containing a joint sequence, and obtains a specific library by PCR amplification.
The technical scheme adopted by the invention is as follows:
a method of constructing a specific library based on Tn7-like transposons, the method comprising the steps of:
(1) Aiming at pathogens to be detected, in a genome conservation area, aiming at more than one conservation sequence, a plurality of specific target DNA with the PAM sequence of NGTN are designed;
specifically, for the conserved sequence 1, a plurality of specific target DNA1 with the PAM sequence of NGTN are designed, wherein the specific target DNA1 is target DNA1-1, target DNA1-2, … and target DNA1-n, and n is an integer more than 2;
designing a plurality of sgRNAs 1 capable of being combined with target DNA1 according to the target DNA1, wherein the sgRNAs 1-1, the sgRNAs 1-2, the sgRNAs … and the sgRNAs 1-n are respectively;
the method can also be designed for a plurality of conserved sequences, and for the conserved sequence 2, a plurality of specific target DNA2 of which the PAM sequence is NGTN is designed, wherein the specific target DNA2 is target DNA2-1, target DNA2-2, … and target DNA2-m, and m is an integer more than 2;
designing a plurality of sgrnas 2 capable of binding to target DNA2 according to target DNA2, wherein the sgrnas 2-1, sgrnas 2-2, … and sgrnas 2-m respectively;
for the conserved sequence t, designing a plurality of specific target DNATs with the PAM sequence of NGTN, wherein the specific target DNATs are target DNAt-1, target DNAt-2, … and target DNAt-p, t is an integer more than 2, and p is an integer more than 2;
designing a plurality of sgRNAs capable of being combined with target DNA according to target DNAt, wherein the sgRNAs are sgRNAt-1, sgRNAt-2, … and sgRNAt-p respectively;
wherein, according to the position of the insert in the sequence, the target DNA comprises a forward target DNA and a reverse target DNA, the 5 'end to the 3' end of the forward target DNA in the guard sequence is PAM-target DNA, the 5 'end to the 3' end of the reverse target DNA in the guard sequence is target DNA-PAM, or the 5 'end to the 3' end of the reverse target DNA in the complementary strand of the guard sequence is PAM-target DNA.
Further, a plurality of target DNAs, such as target DNA1-1, target DNA1-2, …, target DNA1-n, are alternately arranged in the order of 5 'end to 3' end in forward direction target DNA and reverse direction target DNA.
For example, when n=3, 3 target DNA are designed on the conserved sequence 1, then target DNA1-1, target DNA1-2, target DNA1-3 are forward target DNA, reverse target DNA and forward target DNA in order, or reverse target DNA, forward target DNA and reverse target DNA in order.
The pathogen may be a virus, bacterium, fungus, parasite, etc.
(2) The sequence of the insert was designed as follows:
LE-linker A-linker B-RE, and inserting the insert into cloning vector to obtain recombinant vector;
LE is the left end sequence of the Tn7-like transposon, and RE is the right end sequence of the Tn7-like transposon;
the sequence of the LE is preferably TGTACAGTGACAAATT;
the sequence of RE is preferably AGTCACTGTACA.
The linker A and the linker B can adopt common linker sequences of a second generation sequencing library, such as P5 and P7.
(3) In a CRISPR-Cas system constructed by a Tn7-like transposon, the recombinant vector performs a transposition reaction with a nucleic acid sample, and an insertion fragment LE-joint A-joint B-RE is inserted into a conserved sequence of a genome conserved region by utilizing the directional insertion function of Cas 12K; the CRISPR-Cas system comprises a purified Cas12K protein, a tnsB, tnsC, tniQ protein, a plurality of sgrnas, a recombinant vector, and a nucleic acid sample;
the plurality of sgrnas includes a plurality of sgrnas 1, a plurality of sgrnas 2 … …, and a plurality of sgrnas t;
i.e., multiple sgrnas designed for multiple conserved sequences are all in one reaction system.
(4) And (3) carrying out PCR amplification on the nucleic acid containing the inserted fragment to obtain DNA fragments with the two ends respectively being the joint A and the joint B, and purifying an amplified product to obtain a specific library.
In the step (3), a CRISPR-Cas system is constructed by a Tn7-like transposon, and an insertion fragment is inserted into a position 60-66bp downstream of PAM through sgRNA guidance.
Further, the sequence and position of the sgRNA-guided inserts in the guard sequence are different for forward and reverse target DNA;
for forward target DNA, the sequence from the 5 'end to the 3' end is PAM-target DNA, and the insertion position and sequence of the insertion fragment are PAM-target DNA- … -LE-adaptor A-adaptor B-RE;
for reverse target DNA, the sequence from the 5 'end to the 3' end is target DNA-PAM, and the insertion position and sequence of the insertion fragment are RE-linker B-linker A-LE- … -target DNA-PAM;
wherein … represents the insertion sequence being in base spacing with PAM. In general, LE is inserted at a position 60-66bp downstream of PAM.
In the present invention, a plurality of target DNAs, for example, a plurality of target DNA1, target DNA1-2, …, target DNA1-n are alternately arranged in the forward direction and the reverse direction, so that the sequence of inserts is also LE-adaptor A-adaptor B-RE and RE-adaptor B-adaptor A-LE alternately arranged.
In the step (3), the cas12K protein and the tnsB, tnsC, tniQ protein can be obtained by constructing an expression vector containing coding genes of the cas12K protein and the tnsB, tnsC, tniQ protein, transforming genetically engineered bacteria, expressing the cas12K protein and the tnsB, tnsC, tniQ protein, and purifying.
Further, the reaction system of the CRISPR-Cas system is as follows:
cas12K, tnsB, tnsC, tniQ proteins each 50nM,20-100ng nucleic acid sample,100ng of recombinant vector, 600nM each of multiple sgRNAs, 1X reaction Buffer, including 26mM HEPES, 4.2mM TRIS pH8, 50. Mu.g/mL BSA,2mMATP,2.1mM DTT,0.005mM EDTA,0.2mM MgCl 2 28mM NaCl,21mM KCL,1.35% glycerol, 15mM magnesium acetate, pH7.5, was made up to 20. Mu.L with purified water.
The plurality of sgrnas includes a plurality of sgrnas 1, a plurality of sgrnas 2 … …, and a plurality of sgrnas t designed for different conserved sequences;
sgRNA1 includes sgRNA1-1, sgRNA1-2, …, and sgRNA1-n;
sgRNA2 includes sgRNA2-1, sgRNA2-2, …, sgRNA2-m;
the sgRNAs include sgRNAt-1, sgRNAt-2, …, and sgRNAt-p.
And transposing a plurality of sgrnas designed for different conserved sequences in the same reaction system, and inserting the insert LE-adaptor A-adaptor B-RE into the plurality of conserved sequences in a forward and reverse alternating order.
The reaction procedure of the transfer seat reaction in the step (3) is as follows: the reaction was carried out at 42℃for 1 hour and at 85℃for 10 minutes.
In the step (3), the nucleic acid sample is a nucleic acid sample obtained by extracting a sample to be detected.
In the step (4), the PCR amplified primers are a linker A primer and a linker B primer, preferably a linker P5 primer and a linker P7 primer.
In the step (4), the DNA fragments with the two ends respectively being the linker a and the linker B are amplified by PCR amplification of the linker a primer and the linker B primer, and since the inserts are alternately arranged according to LE-linker a-linker B-RE and RE-linker B-linker a-LE in the sequence, the linkers a and the linkers B of the obtained large number of amplified product fragments are respectively located in two adjacent inserts, and for a conserved sequence after transposition, mainly the following four types of fragments are obtained:
linker B-linker A-LE- … … -LE-linker A;
linker A-LE- … … -LE-linker A-linker B;
linker B-RE … … RE-linker B-linker a;
linker A-linker B-RE … … RE-linker B.
After amplification, four main types of fragments are obtained by amplifying each of the conserved sequences containing the insert fragments, and 4*t main fragments can be obtained by t conserved sequences.
Among the amplified product fragments, the adaptor A, B of the adjacent insert fragment contains a great amount of specific sequence information of the nucleic acid sample to be detected, and the amplified product is connected with a sequencing adaptor, so that the amplified product can be directly sequenced on a machine. No further connection of the joint is required.
The invention also provides a method for constructing a specific library to detect pathogens based on Tn7-like transposons, which comprises the following steps: and constructing a specific library according to the method, carrying out second-generation sequencing on the specific library, and carrying out matching analysis on the obtained data and a conserved sequence of a genome of the pathogen to be detected to judge whether the pathogen exists in the sample to be detected.
In one embodiment of the invention, a specific library is constructed for mycobacterium tuberculosis, and detection of tuberculosis pathogens is an application value embodiment of the method (for example, an Illuminate detection platform).
The invention designs a plurality of specific gRNAs close to each other, constructs pDONER plasmids containing the sequences of the joints A and B, constructs a specific library by using a Tn7-like transposon library construction method, and can be used for detecting pathogens.
The invention firstly provides a novel specific pathogen enrichment method by utilizing the activity of inserting a Tn7-like transposon into double-stranded DNA with a short fragment and combining with the directional insertion function of Cas12k, a sequencing joint is inserted into a nucleic acid sample sequence to be detected through a transposition, then the sequencing joint primer is used for amplification, a fragment with the sequencing joint at two ends is obtained through amplification, a specific library is constructed, the specific library is directly used for high-throughput sequencing, a large amount of specific sequence information of the nucleic acid sample is included in the library fragment, and the additional connecting joint is not needed. The invention can detect a plurality of targets, and can judge as a positive sample only by detecting one target sequence, thus enriching target nucleic acid, improving detection sensitivity, particularly collecting more genome information after enriching for pathogens with low load, eliminating false positive influence caused by non-specific PCR amplification and having good application prospect.
Drawings
FIG. 1 is a flow chart of an embodiment of the present invention.
FIG. 2 shows a schematic diagram of the detection of Mycobacterium tuberculosis using a Tn7-like transposon specific library construction method.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified.
Reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
The salient feature of the vector pDonor as template for the insert in the examples described below is the transposon LE and RE sequences flanking the fragment.
EXAMPLE 1 construction of recombinant vector pDONER vector
1. Design and Synthesis of inserts
Synthesizing fragments by using a LE (TGTACAGTGACAAATT) -connector A-connector B-RE (AGTCACTGTACA) sequence structure, and then performing high-fidelity PCR amplification, wherein an amplification reaction system is as follows: 5 XPrimeSTAR Buffer (Mg2+Plus) 10. Mu.L; 1 XdNTP mix (2.5 mM each) 4. Mu.L; primer A, B (10. Mu.M) 1. Mu.L each; primeSTAR HSDNAPolymerase (2.5U/. Mu.L) 0.5. Mu.L; the sterilized water was made up to 50. Mu.L, and the reaction procedure was: pre-denaturing at 95deg.C for 2min;30 cycles (98 ℃ 10s,55 ℃ 5s,72 ℃ 4 min); extending at 72℃for 5min. Then 1% agarose gel electrophoresis is used for detecting the size of the strip, and the gel is cut to recover the fragment as F1, and the concentration is measured for standby.
2. Ligation, transformation and vector validation of fragments
Using the cloning vector pMD 19-T (Takara) as a template, the fragment F1 (40 ng) was mixed and added to a 5. Mu. LTA cloning system, reacted at 16℃for 30 minutes, immediately cooled on ice, and E.coli chemically competent cells for cloning were thawed on ice. Adding 5-10 μl of recombinant product into 100 μl of competent cells, mixing with light elastic tube wall (mixing without shaking), and standing on ice for 30min. After heat shock in a 42 ℃ water bath for 45 seconds, the mixture is immediately placed on ice for cooling for 2 minutes. 900. Mu.L of LB liquid medium (without antibiotics) was added and the mixture was shaken at 37℃for 1h (rotation speed 200 rpm). Ampicillin-resistant LB solid medium plates were pre-heated in an incubator at 37 ℃. Centrifuge at 5,000rpm (2,500 Xg) for 5min and discard 900. Mu.L of supernatant. The bacteria were resuspended in the remaining medium and gently spread on plates containing the correct resistance with sterile spreading bars. After inversion culture for 12-16h in a 37 ℃ incubator, single colony is picked up in ampicillin resistant liquid LB, shaken for 1-2h, positive clone is verified by PCR amplification, and verified bacterial liquid is sequenced by sanger, and no mutation of the sequence is verified.
Example 2, sgRNA design
1. Selection of detection targets and selection of sgrnas
The conserved sequences IS1081 and IS6110 of Mycobacterium tuberculosis were selected for identification of tuberculosis targets.
Designing target DNA and sgRNA for two conserved sequences IS1081 and IS6110 of the mycobacterium tuberculosis respectively, designing 3 specific target DNA1 with the PAM sequence NGTN for the conserved sequence IS1081, namely target DNA1-1, target DNA1-2 and target DNA1-3, and designing a plurality of sgRNAs 1 capable of being combined with the target DNA1 according to the target DNA1, namely sgRNA1-1, sgRNA1-2 and sgRNA1-3 respectively;
for the conserved sequence IS6110, 3 specific target DNA2 with the PAM sequence NGTN are designed, namely target DNA2-1, target DNA2-2 and target DNA2-3, and a plurality of specific sgRNAs 2 which can be combined with the target DNA2 are respectively designed according to the target DNA2, namely sgRNAs, 2-1, sgRNA2-2 and sgRNA2-3;
the conserved sequence IS1081 IS shown below, wherein the grey ground IS PAM, double underlined IS reverse target DNA, single underlined IS forward target DNA
The conserved sequence IS6110 IS shown below, wherein the grey ground IS PAM, double underlined IS reverse target DNA, single underlined IS forward target DNA
Specific sequences of the sgRNAs 1-1, 1-2, 1-3 and 2-1, 2-2 and 2-3 are shown below. The three sequences are used for inserting genes in the forward and reverse directions, and the specific target DNA IS about 150bp in the gene position according to the final PCR amplification size, and the schematic diagram of the sgRNA designed by IS1081 IS shown in FIG. 2. Based on the designed sequence, the sgRNAs 1-1, 1-2, 1-3 and 2-1, 2-2, 2-3 are synthesized.
sgRNA1-1:5’-GGGCUGGUCGUAGAUGGAGU-3’；
sgRNA1-2:5’-CUCGACGCUCUGACCGACAA-3’；
sgRNA1-3:5’-CUUCGAUCCAUUCGUCGUGU-3’；
sgRNA2-1:5’-UGCUACCCACAGCCGGUUAG-3’；
sgRNA2-2:5’-CGCCUACGUGGCCUUUGUCA-3’；
sgRNA2-3:5’-AUAGGAGCUUCCGACCGCUC-3’；
In this example, 3 target DNAs were designed on each conserved sequence, and in the case of IS1081, target DNA1, target DNA2, and target DNA3 were designed, respectively, and corresponding sgRNA1-1, sgRNA1-2, and sgRNA1-3 were designed.
the target DNA1, the target DNA2 and the target DNA3 are reverse target DNA, forward target DNA and reverse target DNA in sequence.
The sequence and the position of the sgRNA guided insert corresponding to the forward target DNA and the reverse target DNA in the conservation sequence are different;
for forward target DNA, the sequence from the 5 'end to the 3' end is PAM-target DNA, and the insertion position and sequence of the insertion fragment are PAM-target DNA- … -LE-adaptor A-adaptor B-RE;
for reverse target DNA, the sequence from the 5 'end to the 3' end is target DNA-PAM, and the insertion position and sequence of the insertion fragment are RE-linker B-linker A-LE- … -target DNA-PAM;
wherein … represents the insertion sequence being in base spacing with PAM. In general, LE is inserted at a position 60-66bp downstream of PAM.
In this example, target DNA1, target DNA2, target DNA3 are reverse target DNA, forward target DNA, and reverse target DNA in this order, and thus the inserted insert sequences are RE-linker B-linker A-LE, LE-linker A-linker B-RE, RE-linker B-linker A-LE.
2. Designing the structure of the inserted DNA fragment: LE (TGTACAGTGACAAATT) -P5 (Illuminate linker sequence) -P7 (Illuminate linker sequence) -RE (AGTCACTGTACA), vectors were constructed and transformed as in steps 1, 2 of example 1 to give the pDONER-MT plasmid.
Example 3 insertion of specific fragments into genome
1. Mycobacterium tuberculosis inactivated sample nucleic acid extraction
After the sample is received, the sample information is checked to extract the nucleic acid without error, and different modes are adopted for different types of samples. Library construction was performed on nucleic acids or dilutions thereof in a total volume of 150-200ng, but not exceeding 35. Mu.L in total, at a library concentration of greater than 500pM. Samples enriched in 1.5mL centrifuge tubes were added to a 2mL shock tube containing 1.2mL LPBS by dipping sesame seed size cells with a 10. Mu.L tip, and the tip was blown and mixed well and operated according to the following standard nucleic acid extraction procedure (Au Cheng Hesuan extraction apparatus Auto-Pure 20B): 1.2mL of the sample to be tested is added into a 2mL vibrating tube in a biosafety cabinet (the sample needs to be balanced to room temperature, if the sample is too thick, 5-10 mu L of the sample can be taken and diluted to 1.2mL by 1 XPBS); the 2mL vibrating tube is placed in a biological sample homogenizer according to SPEED:6.9 (M/S); CYCLE:9, a step of performing the process; TIME:30 (S); INTER:1 (MIN) run. The 2mL shaking tube was taken out, put into a centrifuge, and centrifuged at 12000rpm for 3min. Sequentially adding 20 mu L of proteinase K solution, 1.5mL of lysate and 1mL of pretreated specimen into a No. 1 hole; add 500. Mu.L of cleaning solution I to well number 2; 600 μl of cleaning solution II was added to well number 3; adding 400 mu L of cleaning solution II into a No. 4 hole, extracting 10 mu L of magnetic beads (shaking and mixing for 30s before the magnetic beads are extracted, and shaking and mixing once every two times of magnetic beads are added); 60-100 mu L of eluent is added into the No. 7 hole (the eluent is added at the bottom of the hole and is not added on the hole wall; the reagent-added Auto-Pure20B kit is placed in an O Cheng Hesuan extractor, an Auto-Pure20B magnetic bar sleeve is inserted, a cabin door is closed, a program (see table 1 below) is operated, and a nucleic acid sample is obtained through extraction.
TABLE 1
2. Specific fragment in vitro insertion of genomic nucleic acid
In the presence of ATP and Mg 2+ The buffer system of (2) is added with extracted nucleic acid, various designed sgRNAs, cas12K, tnsB/tnsC/tniQ protein and constructed pDONER-MT plasmid, and the transposition reaction system is as follows:
20. Mu.L of reaction system: cas12K, tnsB/tnsC/tniQ protein (50 nM each), 20-100ng nucleic acid, 100ng pDONER-MT, sgRNA1-1, sgRNA1-2, sgRNA1-3, sgRNA2-1, sgRNA2-2, sgRNA2-3 each 600nM,1 Xresponse Buffer (26 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 4.2mM TRIS pH8, 50. Mu.g/mL BSA,2mM ATP,2.1mM DTT,0.005mM EDTA,0.2mM MgCl 2 28mM NaCl,21mM KCL,1.35% glycerol, 15mM magnesium acetate, pH 7.5), less than 20. Mu.L of purified water. The reaction procedure is: the reaction was carried out at 42℃for 1 hour and at 85℃for 10 minutes.
In this example, 3 sgRNAs were designed for each of the conserved sequences IS1081 and IS6110, and the transposition reaction was performed in one reaction system. Taking the conserved sequence IS1081 as an example, the sequence and position of LE-P5-P7-RE fragment insertions in the transposition reaction under the guidance of 3 sgRNAs IS shown in FIG. 2. CRISPR-Cas inserts the insert into a position 60-60bp downstream of PAM under the guidance of sgRNA.
The sequence and position of insertion of the LE-P5-P7-RE fragment IS similar for the conserved sequence IS 6110.
The inserted nucleic acid sequence comprises
RE-P7-P5-LE-target DNA1-PAM1……PAM2-target DNA2-LE-P5-P7-RE……RE-P7-P5-LE-target DNA3-PAM3
Example 4 fragment amplification and library construction purification
1. Amplifying the inserted nucleic acid with P5 and P7 primers to obtain DNA fragment
P5 primer 5'-AATGATACGGCGACCACCGAGATCTACAC-3';
p7 primer 5'-CAAGCAGAAGACGGCATACGAGAT-3'.
The PCR amplification reaction system is as follows: 5 XPrimeSTAR Buffer (Mg2+Plus) 10. Mu.L; 1 XdNTP mix (2.5 mM each) 4. Mu.L; 1. Mu.L each of the P5 primer and the P7 primer (10. Mu.M); primeSTAR HSDNAPolymerase (2.5U/. Mu.L) 0.5. Mu.L; supplementing sterilized water to 50 mu L, placing the in-vitro prepared PCR tube into a PCR instrument, and reacting according to the following procedures: 30min at 42 ℃;75 ℃ for 10min; hold at 4 ℃; the PCR hotplate temperature was set at 105 ℃. After the reaction was completed, the PCR tube was taken out from the PCR instrument and placed on ice.
Through PCR amplification, P5 and P7 sequences are inserted into nucleic acid, and under the guidance of P5 and P7 primers, DNA fragments with two ends of P5 and P7 are obtained through amplification, and the amplified fragments mainly have four fragment forms by taking a conserved sequence IS1081 as an example, as shown in figure 2.
Comprises P7-P5-LE-target DNA1-PAM1 … … PAM2-target DNA2-LE-P5;
P5-LE-target DNA1-PAM1……PAM2-target DNA2-LE-P5-P7；
P7-RE……RE-P7-P5；
P5-P7-RE……RE-P7。
for the whole reaction system, the conserved sequence IS1081 IS amplified to obtain four main fragment forms, and the same four main fragment forms of the conserved sequence IS6110 are also obtained by amplification, wherein the four main fragment forms are respectively as follows:
P7-P5-LE-target DNA2-1-PAM1……PAM2-target DNA2-2-LE-P5；
P5-LE-target DNA2-1-PAM1……PAM2-target DNA2-2-LE-P5-P7；
P7-RE……RE-P7-P5；
P5-P7-RE……RE-P7。
2. library construction purification
Sucking 40 mu L of the balanced purified magnetic beads into the amplified product solution, and fully vibrating and uniformly mixing. (taking the purified magnetic beads out of the refrigerator at the temperature of 2-8 ℃ in advance, and uniformly mixing the magnetic beads for 30 minutes at room temperature by using a vertical mixer); incubating for 5-10 min at room temperature to enable DNA to be fully bound to the magnetic beads; after instantaneous centrifugation, the reaction tube is placed on a magnetic rack, after the solution is clarified (about 5-10 min), the supernatant liquid is carefully sucked off by a pipette (note that the magnetic beads are not touched); the sample is always placed on a magnetic frame, 200 mu L of purified cleaning solution is added to the reaction tube in the direction far away from the magnetic beads, the reaction tube is incubated at room temperature for 30s, the supernatant is carefully sucked off by a liquid-transferer (note that the magnetic beads are not blown off when the purified cleaning solution is added to rinse the magnetic beads, and the residual supernatant is required to be sucked off as much as possible by using the liquid-transferer after washing); repeating the washing steps once; opening the pipe cover, and airing at room temperature for 1-3 min; the PCR tube was removed from the magnetic rack and 25. Mu.LNuclease-free H was added 2 O or purifying the eluate into a centrifuge tube. Fully vibrating and uniformly mixing, standing for 5min at room temperature, and vibrating and uniformly mixing for 2-3 times in the middle to keep the magnetic beads in a suspension state; after transient centrifugation, the reaction tube is placed on a magnetic rack, after the solution is clarified (about 3-5 min), 23 mu L of supernatant is transferred to a new PCR tube, a mark is made, a specific library is obtained, and on-machine sequencing is performed.
Example 5 interpretation of results
8 fragments containing a large amount of specific sequence information (four fragments of IS1081 are shown in figure 2) obtained by sequencing, and the obtained data are matched with the conserved sequences IS1081 and IS6110 of the genome of the mycobacterium tuberculosis for analysis, so long as one of the matching degrees reaches more than 99%, the detection of the mycobacterium tuberculosis can be considered positive.
According to the invention, a plurality of conserved sequences are transposed and amplified, so that a plurality of targets are detected, and in the data obtained by sequencing a specific library, positive can be judged as long as the matching degree of one amplified fragment and the conserved sequence of a pathogen reaches more than 99%, so that the target nucleic acid can be enriched, the detection sensitivity is improved, and more genome information can be collected after the enrichment is carried out on the pathogen with low load.
Claims (9)
1. A method for constructing a specific library based on Tn7-like transposons, characterized in that the method comprises the steps of:
(1) Aiming at pathogens to be detected, in a genome conservation area, aiming at more than one conservation sequence, a plurality of specific target DNA with the PAM sequence of NGTN are designed;
designing a plurality of sgrnas capable of binding to target DNA according to the target DNA;
the target DNA comprises forward target DNA and reverse target DNA, wherein the position sequence from the 5 'end to the 3' end of the forward target DNA in the guard sequence is PAM-target DNA, and the position sequence from the 5 'end to the 3' end of the reverse target DNA in the guard sequence is target DNA-PAM; a plurality of target DNAs are alternately arranged according to forward target DNA and reverse target DNA in the order from 5 'end to 3' end;
(2) The sequence of the insert was designed as follows:
LE-linker A-linker B-RE, and inserting the insert into cloning vector to obtain recombinant vector;
LE is the left end of the Tn7-like transposon, RE is the right end of the Tn7-like transposon;
(3) In a CRISPR-Cas system constructed by a Tn7-like transposon, the recombinant vector performs a transposition reaction with a nucleic acid sample, and an insertion fragment LE-joint A-joint B-RE is inserted into a conserved sequence of a genome conserved region by utilizing the directional insertion function of Cas 12K; the CRISPR-Cas system comprises a purified Cas12K protein, a tnsB, tnsC, tniQ protein, a plurality of sgrnas, a recombinant vector, and a nucleic acid sample;
(4) And (3) carrying out PCR amplification on the nucleic acid containing the inserted fragment to obtain DNA fragments with the two ends respectively being the joint A and the joint B, and purifying an amplified product to obtain a specific library.
2. The method of claim 1, wherein in the step (1), a plurality of specific target DNA1, wherein the specific target DNA1 is a target DNA1-1, a target DNA1-2, a target DNA …, and a target DNA1-n, wherein n is an integer of 2 or more, are designed for the conserved sequence 1;
designing a plurality of sgRNAs 1 capable of being combined with target DNA1 according to the target DNA1, wherein the sgRNAs 1-1, the sgRNAs 1-2, the sgRNAs … and the sgRNAs 1-n are respectively;
for the conserved sequence 2, designing a plurality of specific target DNA2 with the PAM sequence of NGTN, wherein the specific target DNA2 is target DNA2-1, target DNA2-2, … and target DNA2-m, and m is an integer more than 2;
designing a plurality of sgrnas 2 capable of binding to target DNA2 according to target DNA2, wherein the sgrnas 2-1, sgrnas 2-2, … and sgrnas 2-m respectively;
for the conserved sequence t, designing a plurality of specific target DNATs with the PAM sequence of NGTN, wherein the specific target DNATs are target DNAt-1, target DNAt-2, … and target DNAt-p, t is an integer more than 2, and p is an integer more than 2;
and designing a plurality of sgRNAs capable of combining with target DNA according to target DNAt, wherein the sgRNAs are respectively sgRNAt-1, sgRNAt-2, … and sgRNAt-p.
3. The method of claim 2, wherein in step (1), when n=3, 3 target DNA1 is designed on the conserved sequence 1, including target DNA1-1, target DNA1-2, target DNA1-3, which is sequentially forward target DNA, reverse target DNA, and forward target DNA, or sequentially reverse target DNA, forward target DNA, and reverse target DNA.
4. The method of claim 1, wherein in step (2), the LE has a sequence of TGTACAGTGACAAATT;
the sequence of RE is AGTCACTGTACA.
5. The method of claim 1, wherein in step (3), the Tn7-like transposon constructs a CRISPR-Cas system, and the insert is inserted 60-66bp downstream of the PAM by sgRNA guidance.
6. The method of claim 1, wherein in step (3), the sequence from the 5 'end to the 3' end of the forward target DNA is PAM-target DNA, and the insertion position and sequence of the insert is PAM-target DNA- … -LE-adaptor a-adaptor B-RE;
the sequence from the 5 'end to the 3' end of the reverse target DNA is target DNA-PAM, and the insertion position and sequence of the insertion fragment are RE-adaptor B-adaptor A-LE- … -target DNA-PAM.
7. The method of claim 2, wherein in step (3), the reaction system of the CRISPR-Cas system is:
cas12K, tnsB, tnsC, tniQ protein 50nM each, 20-100ng nucleic acid sample, 100ng recombinant vector, 600nM each of multiple sgRNAs, 1X reaction Buffer, including 26mM HEPES, 4.2mM TRIS pH8, 50 μg/mL BSA,2mMATP,2.1mM DTT,0.005mM EDTA,0.2mM MgCl 2 28mM NaCl,21mM KCL,1.35% glycerol, 15mM magnesium acetate, pH7.5, was made up to 20. Mu.L with purified water; the reaction procedure of the transposition reaction is as follows: reacting for 1h at 42 ℃ and 10 minutes at 85 ℃;
the plurality of sgrnas includes a plurality of sgrnas including a plurality of sgrnas 1, a plurality of sgrnas 2 … ….
8. The method of claim 1, wherein in step (2), the linker a and the linker B are P5 and P7, respectively.
9. The method for constructing a specific library to detect pathogens based on Tn7-like transposons is characterized by comprising the following steps: constructing a specific library according to the method of one of claims 1 to 8, performing second generation sequencing on the specific library, and performing matching analysis on the obtained data and a conserved sequence of a genome of a pathogen to be detected to determine whether the pathogen exists in the sample to be detected.
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