CN111718987A - SLCO1B1 gene detection primer group, detection kit and detection method - Google Patents
SLCO1B1 gene detection primer group, detection kit and detection method Download PDFInfo
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention provides an SLCO1B1 gene detection primer group, a detection kit and a detection method, comprising an rs2306283 locus amplification primer pair SEQ ID NO. 1-2; wherein the amplification primers comprise a cleavable site or a cleavable sequence. The invention designs the primer group, directly detects the SNP locus to be detected by a high-throughput sequencing method, avoids the complex analysis after sequencing and provides a simple and convenient method for detecting the locus by second-generation sequencing.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to an SLCO1B1 gene detection primer group, a detection kit and a detection method.
Background
The SLCO1B1 encoded product in human body is organic anion transport polypeptide, and is an uptake type transport protein specifically expressed on the surface of liver cells. SLCO1B1-388A > G was first found to result in a decrease in pravastatin bioavailability. Research shows that 388GG allele can increase the lipid-lowering effect of statins.
At present, the detection of gene mutation and gene polymorphism is commonly carried out by Sanger sequencing method, gene chip hybridization method, Taqman fluorescence probe method, allele specific Amplification method (Amplification purification System Real Time PCR, ARMS-RT PCR) and the like.
The sanger sequencing method has accurate result, but has more detection steps, complicated operation and lower detection sensitivity; the gene chip hybridization method has more detection steps, complicated operation, lower detection sensitivity and the problem of false positive; the Taqman fluorescent probe method and the ARMS-RT PCR method have low detection flux.
The influence of SLCO1B1c.388A > G polymorphism on the effects of atorvastatin lipid reduction and atherosclerosis resistance of a Chinese ischemic stroke patient, Wu Yinyan and the like, the nationality of cerebrovascular disease, 2017 (25): 33-38 discloses a method for detecting SLCO1B1 polymorphism, amplifying fragments containing related sites in genome, and performing genotyping detection by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). But the method has lower detection flux and higher cost.
Therefore, it is important to provide a method for detecting the polymorphism of SLCO1B1, which has high sensitivity, short detection cycle, high detection flux, low cost and low false positive rate.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides an SLCO1B1 gene detection primer group, a detection kit and a detection method, and the detection method has the advantages of high sensitivity, short detection period, high detection flux and low cost aiming at the gene type of the rs2306283 locus of the SLCO1B1 gene.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer group for detecting an SLCO1B1 gene, which comprises an amplification primer pair SEQ ID NO.1-2 at the rs2306283 site; wherein the amplification primers comprise a cleavable site or a cleavable sequence.
The sequence of the upstream primer of the amplification primer is SEQ ID NO. 1:
5’-ACGGUGCATAATGCCTGGAATACAGTA-3’.
the sequence of the downstream primer of the amplification primer is SEQ ID NO. 2:
5’-TGTCAGATATTCTTACCGGCAG-3’.
second generation sequencing (high throughput sequencing) a sequencing library was constructed by randomly disrupting the gene/genome sequence, and the library was then sequenced to obtain the sequence. And comparing/splicing the deep sequencing sequence, comparing and analyzing the deep sequencing sequence with a reference genome, and carrying out bioinformatics analysis, so as to obtain the genotype of the site to be detected. The analysis difficulty after sequencing is higher, and false negative/false positive problems exist. The invention designs the primer group, directly detects the SNP locus to be detected by a high-throughput sequencing method, avoids the complex analysis after sequencing and provides a simple and convenient method for detecting the locus by second-generation sequencing.
In a second aspect, the present invention provides a detection kit for the SLCO1B1 gene, wherein the kit comprises the primer set of the first aspect.
Preferably, the kit further comprises a linker, a cleavage agent that specifically cleaves the cleavable site or cleavable sequence of the amplification primer, a ligase, and a ligase buffer.
Preferably, the adaptor also has a tag sequence thereon.
In the present invention, in order to distinguish different sample sources during the simultaneous detection process, the tag sequence is designed on the adaptor in this embodiment, and may be composed of 1-14 bases (A, T, C or G), and the tag sequence is selected according to the number of samples, so as to facilitate the analysis of the sample source corresponding to the detection result.
Preferably, the linker is linked to the amplification product containing the rs2306283 site which has been subjected to cleavage treatment.
Preferably, the adaptor is a double-stranded double-protruding-end adaptor or a single-stranded linker.
Preferably, the nucleotide sequence of one strand of the double-stranded double-overhang end linker is shown as SEQ ID No.3, the nucleotide sequence of the other strand is shown as SEQ ID No.4, and the specific sequences are shown as follows:
SEQ ID NO.3：
CCTCCCTGCAGTCTCTATGGGCNNNNCTGCTAGTCGCTGAAGTAGTCGGT.
SEQ ID NO.4：
CTACTTCAGCGACTAGCAGNNNNGCCCATAGAGACTGCAGGG.
preferably, the nucleotide sequence of the single-chain linker is shown as SEQ ID NO. 5.
SEQ ID NO.5：
CCTCCCTGCAGTCTCTATGGGCNNNNCTGCTAGTCGCTGAAGTAGAGCATAATGC.
Preferably, the cleavage agent comprises a USER enzyme.
Preferably, the kit further comprises a site anchor sequence SEQ ID NO.6 and a tag anchor sequence SEQ ID NO. 7.
SEQ ID NO.6：GATATTAGTTTCTTTAGA.
SEQ ID NO.7：GCCCATAGAGACT.
In a third aspect, the present invention provides a method for detecting the SLCO1B1 gene for the purpose of non-disease diagnosis and treatment, wherein the method comprises the following steps:
(1) amplifying a gene fragment containing the rs2306283 site of the SLCO1B1 gene by using an amplification primer, connecting an amplification product with a joint to construct a library to be detected, fixing library molecules on a solid phase carrier in an addressable manner, and denaturing the fragment to be detected into a single chain;
(2) connecting the site anchoring sequence to a library molecule to be detected on a solid phase carrier, adding a fluorescence-labeled oligonucleotide probe for a connection reaction, and performing denaturation to remove a connection sequencing product;
(3) adding a label anchoring sequence of a label on the detection joint, adding a fluorescence-labeled oligonucleotide probe, performing ligation reaction, performing positioning addressing on the library, and performing denaturation to remove a ligation sequencing product.
Preferably, the method for immobilizing on a solid support in step (1) comprises: connecting the amplification product subjected to the fracture treatment with a joint, wherein the joint is fixed on the surface of magnetic beads, spotting the magnetic beads onto a glass slide for fixation, or performing secondary amplification on the amplification product of the first PCR by using a primer fixed on a solid phase carrier through secondary PCR amplification, and fixing the amplification product of the first PCR on the solid phase carrier.
Preferably, the linker has a biotin modification at one end.
Preferably, the magnetic beads carry a streptavidin modification.
Preferably, the slide is isothiocyanato modified.
Preferably, the immobilization time is 35-40 ℃, for example 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ or 40 ℃, preferably 37 ℃.
Preferably, the fixed time is 0.5-1.5h, for example 0.5, 0.8, 1, 1.2, 1.5h, preferably 1 h.
Preferably, the method specifically comprises the following steps:
(1) amplifying a gene fragment containing the rs2306283 site of the SLCO1B1 gene by using an amplification primer, breaking an amplification product, connecting the amplification product with a joint to construct a library to be detected, fixing library molecules on a solid phase carrier in an addressable manner, and denaturing the fragment to be detected into a single chain;
(2) connecting the site anchoring sequence to a library molecule to be detected on a solid phase carrier, adding a fluorescence-labeled oligonucleotide probe, ligase and a ligase buffer solution, carrying out a ligation reaction, and denaturing to remove a ligation sequencing product;
(3) adding a label anchoring sequence on the detection joint, adding a fluorescence-labeled oligonucleotide probe, ligase and ligase buffer solution, carrying out ligation reaction, carrying out positioning addressing on the library, and denaturing to remove a ligation sequencing product.
The detection process of the invention is as follows:
1. amplifying the genomic DNA with primers (the amplification product comprises rs2306283 site), the amplification primers used containing cleavable sites (for cleavage of the PCR amplification product there, followed by ligation with an adaptor);
2. breaking the PCR product at the break point of the primer by an enzyme digestion or chemical treatment method;
3. connecting the broken amplification product with a joint to construct a sequencing library;
4. connecting the sequencing library to a solid phase carrier, and denaturing the fragment to be detected into a single chain.
5. Anchoring an anchoring primer for detecting the rs2306283 site on a library molecule to be detected connected to a solid phase carrier; adding a fluorescent-labeled oligonucleotide probe, ligase and a corresponding buffer solution to perform a ligation reaction; denaturing to remove the ligation sequencing product;
6. adding an anchor primer of a label on a detection joint and anchoring the anchor primer on the joint of the library to be detected; adding a fluorescent-labeled oligonucleotide probe, ligase and a corresponding buffer solution to perform a ligation reaction; the library is addressed positionally. Denaturation removes the ligation sequencing product.
The method has the advantages of high sensitivity, short detection period, high detection flux and low cost aiming at SLCO1B1 gene rs2306283 locus genotype.
Compared with the prior art, the invention has the following beneficial effects:
(1) the primer group for detecting the SLCO1B1 gene has good specificity and high sensitivity, and can effectively carry out typing on the SLCO1B1 gene;
(2) the kit provided by the invention is used for detecting SLCO1B1 genotyping specifically, has high specificity and good sensitivity, obviously improves the detection flux, directly detects the target site, has high SNP sequencing depth (10000 sequencing depth) of the target site, high accuracy and high sensitivity, does not need bioinformatics analysis, and only detects the target site.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following embodiments further illustrate the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments.
Example 1 SLCO1B1 genotyping assay
1. PCR amplification
Human blood genome DNA is used as a template, and a sequence of an SNP site is amplified by using a site amplification primer pair SEQ ID NO.1-2, wherein the reaction system comprises an F primer (10 mu.M), 1 mu.L, an R primer (10 mu.M), 1 mu.L, dNTPs (2.5 mM each), 4 mu.L, blood genome DNA, 50ng, HotStart Taq (5U/mu.L), 0.5 mu.L, 10 × HotStart Taq Buffer, 5 mu.L and ddH2O was added to 50. mu.L.
The PCR reaction conditions were as follows: 10min at 95 ℃; 95 ℃ for 15s, 56 ℃ for 30s, and 72 ℃ for 50 s; repeat 30 cycles; 3min at 72 ℃. The obtained product is the PCR amplification product.
Detecting through agarose gel electrophoresis, wherein the first amplification products of all samples contain target molecules, and a gel electrophoresis picture shows that the sample has no miscellaneous bands and a single band; thus indicating that the PCR amplification successfully obtains the desired PCR amplification product.
2. Library molecule construction
In order to distinguish different sample sources in the simultaneous detection process, the present embodiment designs a tag sequence on the adaptor.
The double-stranded double-protruding-end linker SEQ ID NO.3-4 was bound to streptavidin-modified Myone magnetic beads (Invitrogen) such that each of the linkers was immobilized on the surface of the magnetic beads, and the reaction system and the reaction process were performed by binding 200ng of the linker to 4. mu.L (about 4 × 10)7Magnetic beads) Myone magnetic beads were mixed by spiral shaking for 30min, and mixed with a suitable amount of TE buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA) was washed twice, centrifuged, and the resulting magnetic beads were washed with 4 μ L of binding buffer (10mM Tris-HCl, ph 7.5; 1mM EDTA; 1M NaCl; 0.01% Triton X-100) to obtain magnetic beads immobilized with double-stranded double-protruding-end linkers.
3. Ligation of PCR product to adaptor
20 mu L of PCR amplification product; USER enzyme (1U/. mu.L, NEB, Cat # M5505S), 10. mu.L; buffer, 8 μ L; t4DNA ligase, 2. mu.L; 0.4 mu L of magnetic beads fixed with double-chain double-protrusion end joints; add ddH2O to 40. mu.L.
Wherein the buffer solution contains 400mM Tris and 100mM MgCl2100mM DTT, 5mM ATP, 25% PEG6000, pH 7.8.
And reacting for 20 minutes at 25 ℃ to obtain a connecting product, namely the sequency library molecule to be detected.
After the reaction is finished, combining 50 tubes of connecting products into 1 tube, centrifuging for 3min at 2500g, adsorbing magnetic beads by a magnet, removing supernatant, washing twice by 50 mu L of TE, and finally suspending in 50 mu L of TE, thereby fixing the library molecules to be detected on the magnetic beads.
Optionally, the attaching of the PCR product to the magnetic bead can be performed by:
the single-chain linker SEQ ID NO.5 was bound to streptavidin-modified Myone magnetic beads (Invitrogen), respectively, so thatThe single-chain linker was immobilized on the surface of the magnetic beads, and the reaction system and the reaction process were carried out by mixing 200ng of the single-chain linker with 4. mu.L (about 4 × 10)7Magnetic beads) Myone magnetic beads were mixed by spiral shaking, reacted for 30min, and then diluted with an appropriate amount of TE buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA) was washed twice, centrifuged, and the resulting magnetic beads were washed with 4 μ L of binding buffer (10mM tris-HCl, ph 7.5; 1mM EDTA; 1M NaCl; 0.01% Triton X-100) to obtain magnetic beads immobilized with single-chain linkers.
The reaction system is magnetic beads with single-chain linker, 5 × 104R primer (10. mu.M), 2. mu.L, dNTPs (2.5 mM each), 4. mu.L, first PCR amplification product, 20ng, HotStart Taq (5U/. mu.L), 0.5. mu.L, 10 × HotStart Taq Buffer, 5. mu.L, ddH2O to 50. mu.L.
The PCR reaction conditions were as follows: 10min at 95 ℃; 95 ℃ for 15s, 56 ℃ for 20s, and 72 ℃ for 50 s; repeat 30 cycles; 3min at 72 ℃.
4. Addressable immobilization of library molecules to be sequenced immobilized on magnetic beads.
And (3) spotting the product obtained in the step (3) to an isothiocyanic modified sample carrying sheet (glass slide), fixing for 1h at 37 ℃, completing addressable fixing of library molecules to be sequenced fixed on magnetic beads, and denaturing the fragments to be sequenced into single chains.
Wherein, the sequencing platform: the high-throughput gene sequencer Pstar II A sequencer of Shenzhen Huaxingkang gene science and technology Limited and the sequencing is carried out by adopting a connection sequencing method.
And (3) sequencing: the sequencing library immobilized on the magnetic beads was added to a sequencing chip and loaded into a sequencer.
The site anchor sequence is fixed and the site genotype is determined by adding a sequencing probe.
The tag anchor sequence is immobilized and tag information is determined by the addition of a sequencing probe.
In conclusion, the detection kit and the detection method of the invention realize high specificity and high sensitivity detection through the primer group designed specifically, effectively carry out typing on the SLCO1B1 gene, have high detection flux, low cost and short period, are different from the existing second-generation sequencing method, directly detect the target locus, have high SNP sequencing depth (10000 sequencing depth) of the target locus, have high accuracy and high sensitivity, do not need bioinformatics analysis, and only detect the target locus.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> grand fugaxian commemorative hospital of Zhongshan university; shenzhen Huaxinkang Gene science and technology Limited
<120> SLCO1B1 gene detection primer group, detection kit and detection method
<130>2019
<160>7
<170>PatentIn version 3.3
<210>1
<211>27
<212>DNA
<213> artificially synthesized sequence
<400>1
acggugcata atgcctggaa tacagta 27
<210>2
<211>22
<212>DNA
<213> artificially synthesized sequence
<400>2
tgtcagatat tcttaccggc ag 22
<210>3
<211>50
<212>DNA
<213> artificially synthesized sequence
<220>
<221>misc_feature
<222>(23)..(26)
<223>n is a, c, g, or t
<400>3
cctccctgca gtctctatgg gcnnnnctgc tagtcgctga agtagtcggt 50
<210>4
<211>42
<212>DNA
<213> artificially synthesized sequence
<220>
<221>misc_feature
<222>(20)..(23)
<223>n is a, c, g, or t
<400>4
ctacttcagc gactagcagn nnngcccata gagactgcag gg 42
<210>5
<211>55
<212>DNA
<213> artificially synthesized sequence
<220>
<221>misc_feature
<222>(23)..(26)
<223>n is a, c, g, or t
<400>5
cctccctgca gtctctatgg gcnnnnctgc tagtcgctga agtagagcat aatgc 55
<210>6
<211>18
<212>DNA
<213> artificially synthesized sequence
<400>6
gatattagtt tctttaga 18
<210>7
<211>13
<212>DNA
<213> artificially synthesized sequence
<400>7
gcccatagag act 13
Claims (10)
1. A primer group for detecting SLCO1B1 gene is characterized by comprising an amplification primer pair SEQ ID NO.1-2 of rs2306283 locus; wherein the amplification primers comprise a cleavable site or a cleavable sequence.
2. An SLCO1B1 gene detection kit, which comprises the primer set of claim 1.
3. The kit of claim 2, further comprising a linker, a cleavage agent that specifically cleaves the cleavable site or cleavable sequence of the amplification primer, a ligase, and a ligase buffer.
4. The kit of claim 3, wherein the adaptor further comprises a tag sequence;
preferably, the linker is directly linked to the amplification product comprising the rs2306283 site.
5. The kit of claim 3 or 4, wherein the adaptor is a double stranded double protruding end adaptor or a single linker.
6. The kit according to claim 5, wherein the nucleotide sequence of one strand of the double-stranded double-overhang end-linker is shown as SEQ ID No.3, and the nucleotide sequence of the other strand is shown as SEQ ID No. 4;
preferably, the nucleotide sequence of the single-chain linker is shown as SEQ ID NO. 5.
7. The kit of any one of claims 3 to 6, wherein the cleavage agent comprises a USER enzyme;
preferably, the kit further comprises a site anchor sequence SEQ ID NO.6 and a tag anchor sequence SEQ ID NO. 7.
8. A method for detecting the SLCO1B1 gene for the purpose of non-disease diagnosis and treatment, wherein the detection is performed by using the primer set of claim 1 or the kit of any one of claims 2 to 7, and the detection method comprises the following steps:
(1) amplifying a gene fragment containing the rs2306283 site of the SLCO1B1 gene by using an amplification primer, connecting an amplification product with a joint to construct a library to be detected, fixing library molecules on a solid phase carrier in an addressable manner, and denaturing the fragment to be detected into a single chain;
(2) connecting the site anchoring sequence to a library molecule to be detected on a solid phase carrier, adding a fluorescence-labeled oligonucleotide probe for a connection reaction, and performing denaturation to remove a connection sequencing product;
(3) adding a label anchoring sequence on the detection joint, adding a fluorescence labeled oligonucleotide probe, carrying out a ligation reaction, carrying out positioning addressing on the library, and carrying out denaturation to remove a ligation sequencing product.
9. The method of claim 8, wherein the immobilization on the solid support of step (1) comprises: connecting the amplification product with a joint, wherein the joint is fixed on the surface of magnetic beads, spotting the magnetic beads onto a glass slide for fixation or amplifying by secondary PCR, performing secondary amplification on the amplification product of the first PCR by using a primer fixed on a solid phase carrier, and fixing the amplification product of the first PCR on the solid phase carrier;
preferably, one end of the linker is modified by biotin;
preferably, the magnetic beads carry streptavidin modifications;
preferably, the slide is isothiocyanato modified;
preferably, the fixed time is 35-40 ℃, preferably 37 ℃;
preferably, the fixed time is 0.5 to 1.5h, preferably 1 h.
10. The method according to claim 8 or 9, characterized in that it comprises in particular the steps of:
(1) amplifying a gene fragment containing the rs2306283 site of the SLCO1B1 gene by using an amplification primer, breaking an amplification product, connecting the amplification product with a joint to construct a library to be detected, fixing library molecules on a solid phase carrier in an addressable manner, and denaturing the fragment to be detected into a single chain;
(2) connecting the site anchoring sequence to a library molecule to be detected on a solid phase carrier, adding a fluorescence-labeled oligonucleotide probe, ligase and a ligase buffer solution, carrying out a ligation reaction, and denaturing to remove a ligation sequencing product;
(3) adding a label anchoring sequence on the detection joint, adding a fluorescence-labeled oligonucleotide probe, ligase and ligase buffer solution, carrying out ligation reaction, carrying out positioning addressing on the library, and denaturing to remove a ligation sequencing product.
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Application publication date: 20200929 |