CN108949967B

CN108949967B - Specific primers and kits for detection of drug gene polymorphisms in cardiovascular diseases by liquid chip technology

Info

Publication number: CN108949967B
Application number: CN201810977398.2A
Authority: CN
Inventors: 付文金
Original assignee: DONGGUAN HOUJIE HOSPITAL
Current assignee: DONGGUAN HOUJIE HOSPITAL
Priority date: 2018-08-23
Filing date: 2018-08-23
Publication date: 2022-03-11
Anticipated expiration: 2038-08-23
Also published as: CN108949967A

Abstract

The invention discloses a primer set for simultaneously detecting 15 kinds of cardiovascular drug-related target gene polymorphisms by using liquid phase chip technology, including 15 sets of primers, wherein each set of primers includes a set of primers for detecting a target gene polymorphism A pair of specific amplification primers and two ASPE primers for the wild type and mutant type of the target gene, respectively. The present invention also discloses a kit comprising these primer sets and a method of using the same. The primer set and the kit of the invention can amplify the target sequence in a one-step PCR reaction, and combine with MagPlex microspheres to simultaneously detect 15 kinds of common cardiovascular drugs-related target gene polymorphisms, with high detection throughput and high cost performance. The detection can provide medication guidance for patients with cardiovascular disease, and provide laboratory basis for clinical rational drug use; the kit of the present invention has very good sensitivity, stability and accuracy in practical applications, and is in good agreement with the gene sequencing results, and has a large Reduced inspection costs.

Description

Specific primer and kit for detecting cardiovascular disease drug gene polymorphism by liquid phase chip technology

Technical Field

The invention relates to the fields of biotechnology and medicine, in particular to specific primers for detecting gene polymorphism of common cardiovascular disease drugs by using a liquid chip technology, and also relates to a kit containing the specific primers and a kit using method.

Background

Cardiovascular disease is caused by heart and vascular disorders, including coronary heart disease (heart attack), cerebrovascular disease (stroke), hypertension (elevated blood pressure), peripheral vascular disease, rheumatic heart disease, congenital heart disease, heart failure, and cardiomyopathy. Cardiovascular disease is a common disease that seriously threatens the health of humans, particularly the middle aged and elderly people over 50 years old. At present, cardiovascular disease death accounts for the first cause of total death of urban and rural residents, 45.01% in rural areas and 42.61% in cities. Statistically, about one third does not die from the disease itself, but from improper medication. Accurate individual administration of cardiovascular diseases is the guarantee of safe administration, so that doctors can individually select the types and the dosage of the medicines for patients.

The clinical common cardiovascular disease treatment drugs comprise clopidogrel, warfarin, nitroglycerin, aspirin and the like. Due to the individual differences, different patients take the same drug with different therapeutic effects. More and more researches show that genetic factors of patients are important factors influencing drug metabolism, absorption and excretion, and at present, dozens of enzymes have been detected to have different activities in different individuals, and as Single Nucleotide Polymorphisms (SNPs) of important genes related to drug absorption, distribution, metabolism and excretion, such as CYP2C9, VKORC1, CYP4F2, CYP2C19, PON1, CYP2D6, ADRB1, ALDH2, GP1BA, PEAR1 and AGTR1, are successively discovered, the determination of drug selection or dosage by detecting the polymorphism of target genes of various cardiovascular drugs is a crucial means for assisting clinical precision medication.

Relevance of CYP2C9 gene, VKORC1 gene and CYP4F2 gene polymorphism and warfarin personalized medicine

Warfarin (warfarin) is a coumarin oral anticoagulant drug, is one of the most widely clinically applied oral anticoagulant drugs at present, but has great difference between clinical curative effect and adverse reaction individuals and difficult mastering of dosage. The research shows that the gene polymorphism of vitamin K epoxide reductase complex subunit I gene (VKORC1) and cytochrome P450 gene (CYP2C9) are the two most main genetic factors influencing the individual difference of warfarin dosage. In addition, the polymorphism of CYP4F2 gene (rs2108622) is also related to individual dose differences of warfarin.

Relevance of CYP2C19 gene polymorphism and clopidogrel personalized medicine

Clopidogrel (Clopidogrel), an oral antiplatelet drug, has been widely used for Acute Coronary Syndrome (ACS) and PCI post-operative treatment at present, and can effectively prevent the occurrence of ischemic time in ACS patients. The medicine can irreversibly combine with platelet surface ADP receptor P2Y12 after in vivo P450 enzyme biotransformation, thereby inhibiting platelet aggregation induced by ADP and achieving anticoagulation effect. Therefore, the cytochrome P450 gene (CYP2C19) is the most important genetic factor influencing individual difference of the dose of clopidogrel.

Relevance of ALDH2 gene polymorphism and nitroglycerin personalized drug administration-

ALDH2 is a main way for catalyzing the biotransformation of nitroglycerin by human bodies, and the enzyme has catalytic activities of dehydrogenase and esterase, wherein the esterase activity can generate NO by denitrifying nitroglycerin, thereby causing vasodilatation. The ALDH2 gene is located at 12q24.2 position of chromosome 12, has the total length of 43,438bp, has 13 exons, encodes polypeptide consisting of 517 amino acid residues, and has 3 cases in the genotype of the gene in the population due to inheritance, and has a homozygote type with normal catalytic activity: ALDH2 x 1/' 1; heterozygote type with reduced catalytic activity: ALDH2 x 2/' 1; homozygote type with loss of catalytic activity: ALDH2 x 2/'2'. Research in recent years shows that ALDH2 can improve the prognosis of heart failure, protect myocardial ischemia-reperfusion injury, cause enzyme activity reduction after gene mutation, and increase the risk of coronary heart disease (CAD) and Myocardial Infarction (MI).

Correlation of polymorphism of CYP2D6, ADRB1, PON1, GP1BA, PEAR1 and AGTR1 genes with cardiovascular disease drugs

The polymorphism of CYP2D6 and ADRB1 genes is related to the curative effect of beta receptor blockers, the polymorphism (rs662) of PON1 gene is related to the curative effect or adverse reaction of clopidogrel, the polymorphism (rs2243093) of GP1BA gene is related to the drug effect of aspirin on antiplatelet, PEAR1 is related to aspirin resistance and prognosis, and the polymorphism (rs5186) of AGTR1 gene is related to hypertension.

In the prior art, a PCR-direct sequencing method, a PCR-pyrophosphoric acid sequencing method, a fluorescent quantitative PCR method, a PCR-high resolution melting curve method, an allele specific PCR method, a PCR-restriction fragment length polymorphism method, a solid chip method and the like are often adopted to detect the polymorphism of one or more cardiovascular drug related genes, the methods have low flexibility, high cost, low flux, long time, expensive detection instruments and are not suitable for clinical popularization, the cost of a reagent for one-time examination of each person reaches dozens to hundreds of dollars, and the existing detection kit does not aim at the Chinese genetic disease spectrum. Therefore, there is a great need for products that specifically detect polymorphisms of a plurality of cardiovascular drug-related genes simultaneously or in parallel.

Disclosure of Invention

Aiming at the defects in the prior art, the inventor of the invention combines the Luminex liquid chip technology with Allele Specific Primer Extension (ASPE) reaction, designs and constructs a simulation sample containing CYP2C9, VKORC1, CYP4F2, CYP2C19, PON1, CYP2D6, ADRB1, ALDH2, GP1BA, PEAR1 and AGTR1 gene mutation sites, and establishes a novel detection method capable of simultaneously detecting the polymorphism of cardiovascular drug related genes.

The invention provides a primer group for simultaneously detecting 15 cardiovascular drug related target gene polymorphisms by using a liquid chip technology, which comprises 15 groups of primers, wherein each group of primers comprises a pair of specific amplification primers for detecting one target gene polymorphism and two ASPE primers respectively aiming at a wild type and a mutant type of the target gene;

the detected target gene polymorphisms were: CYP2C9-C430T rs1799853, CYP2C9-A1075C rs1057910, VKORC1-G1639A rs9923231, CYP4F2-C1347T rs2108622, CYP2C19-G681A rs4244285, CYP2C19-G636 rs4986893, CYP2C19-C806 19 rs 48560, PON 19-Q192 19 rs662, CYP2D 19-C2850 19 rs 947, CYP2D 19-C100 19 rs1065852, ADRB 19-G1165 rs1801253, ALDH 19-G19 rs671, GP1 19-T5 19 rs 22422422422493, PEAR 72G > Ars 27759 and AGTR 1163672-5172A 515186;

the sequences of the primer sets for detecting the site polymorphism of the target gene are as follows:

specific amplification primers for detecting CYP2C9-C430T rs1799853 are shown as sequences SEQ ID NO.1 and SEQ ID NO.2, and ASPE primers are shown as sequences SEQ ID NO.3 and SEQ ID NO. 4;

specific amplification primers for detecting CYP2C9-A1075C rs1057910 are shown as sequences SEQ ID NO.5 and SEQ ID NO.6, and ASPE primers are shown as sequence SEQ ID NO.7 and sequence SEQ ID NO. 8;

specific amplification primers for detecting VKORC1-G1639A rs9923231 are shown as sequences SEQ ID NO.9 and SEQ ID NO.10, and ASPE primers are shown as sequences SEQ ID NO.11 and SEQ ID NO. 12;

specific amplification primers for detecting CYP4F2-C1347T rs2108622 are shown as sequences SEQ ID NO.13 and SEQ ID NO.14, and ASPE primers are shown as sequences SEQ ID NO.15 and SEQ ID NO. 16;

specific amplification primers for detecting CYP2C19-G681Ars4244285 are shown as sequences SEQ ID NO.17 and SEQ ID NO.18, and ASPE primers are shown as sequences SEQ ID NO.19 and SEQ ID NO. 20;

specific amplification primers for detecting CYP2C19-G636A rs4986893 are shown as sequences SEQ ID NO.21 and SEQ ID NO.22, and ASPE primers are shown as sequences SEQ ID NO.23 and SEQ ID NO. 24;

specific amplification primers for detecting CYP2C19-C806T rs12248560 are shown as sequences SEQ ID NO.25 and SEQ ID NO.26, and ASPE primers are shown as sequences SEQ ID NO.27 and SEQ ID NO. 28;

specific amplification primers for detecting PON1-Q192R (A/G) rs662 are shown as sequences SEQ ID NO.29 and SEQ ID NO.30, and ASPE primers are shown as sequences SEQ ID NO.31 and SEQ ID NO. 32;

specific amplification primers for detecting CYP2D6-C2850T rs16947 are shown as sequences SEQ ID NO.33 and SEQ ID NO.34, and ASPE primers are shown as sequences SEQ ID NO.35 and SEQ ID NO. 36;

specific amplification primers for detecting CYP2D6-C100T rs1065852 are shown as sequences SEQ ID NO.37 and SEQ ID NO.38, and ASPE primers are shown as sequences SEQ ID NO.39 and SEQ ID NO. 40;

specific amplification primers for detecting ADRB1-G1165C rs1801253 are shown as sequences SEQ ID NO.41 and SEQ ID NO.42, and ASPE primers are shown as sequences SEQ ID NO.43 and SEQ ID NO. 44;

specific amplification primers for detecting ALDH2-G504A rs671 are shown as sequences SEQ ID NO.45 and SEQ ID NO.46, and ASPE primers are shown as sequences SEQ ID NO.47 and SEQ ID NO. 48;

specific amplification primers for detecting GP1BA-T5C rs2243093 are shown as sequences SEQ ID NO.49 and SEQ ID NO.50, and ASPE primers are shown as sequences SEQ ID NO.51 and SEQ ID NO. 52;

specific amplification primers for detecting that PEAR 1G is greater than A rs2768759 are shown as sequences SEQ ID NO.53 and SEQ ID NO.54, and ASPE primers are shown as sequences SEQ ID NO.55 and SEQ ID NO. 56;

the specific amplification primers for detecting AGTR1-A1166C rs5186 are shown as sequences SEQ ID NO.57 and SEQ ID NO.58, and the ASPE primers are shown as sequences SEQ ID NO.59 and SEQ ID NO. 60.

In a preferred embodiment of the present invention, the ASPE primer comprises two parts, a Tag sequence at 5 ' end and a specific primer sequence for target gene polymorphism at 3 ' end, and the first base at 3 ' end of the ASPE primer is complementary paired with the target gene to be detected.

In another preferred embodiment of the present invention, the specific primer sequence included in the ASPE primer is shown as sequence SEQ ID NO. 61-90.

The second aspect of the invention provides the application of the primer group in the preparation of a kit for simultaneously detecting the polymorphism of 15 cardiovascular drug-related target genes by using a liquid chip technology.

The third aspect of the present invention provides a kit for simultaneously detecting polymorphisms of a plurality of cardiovascular drug-related target genes using a liquid chip technology, the kit comprising the primer set of claim 1 and 30 microspheres, wherein,

the primer group comprises 15 groups of primers, each group of primers comprises a pair of specific amplification primers for detecting polymorphism of a target gene and two ASPE primers respectively aiming at wild type and mutant type of the target gene, the ASPE primers comprise a Tag sequence positioned at a 5 ' end and a specific primer sequence positioned at a 3 ' end and aiming at the target gene, and a first base at the 3 ' end of the ASPE primers is complementarily paired with the target gene to be detected; and

each microsphere is coupled with an addressing probe sequence which is respectively in complementary pairing with the Tag sequence of the ASPE primer.

In a preferred embodiment of the present invention, the kit of the present invention further comprises a streptavidin-phycoerythrin hybridization buffer, a PCR reaction solution, a DNA polymerase and a negative control.

In another preferred embodiment of the present invention, the specific primer sequence at the 3' end included in the ASPE primers in the kit of the present invention is represented by the sequence SEQ ID NO. 61-90.

In yet another preferred embodiment of the present invention, the negative control in the kit of the present invention is distilled water plus microspheres.

A third aspect of the invention provides a method of using the kit of the invention, the method comprising the steps of:

(1) multiplex PCR amplification reaction: extracting DNA in a sample as a template, preparing a multiple PCR reaction system by using a specific amplification primer in the kit, and performing PCR amplification under a set PCR amplification program;

(2) purification of PCR products: digesting redundant dNTP, primers and single-stranded products in the PCR product by using exonuclease and alkaline phosphatase;

(3) ASPE reaction: carrying out extension reaction on the purified PCR product under the designed ASPE reaction program and under the action of DNA polymerase in an ASPE reaction system when the first base at the 3' end of the primer is complementary with the amplified target sequence detection site, and stopping the extension reaction if the first base is not complementary;

(4) and (3) hybridization reaction: taking 25 mu L of diluted microspheres to a 96-well hybridization plate, adding microspheres and deionized water with the same amount into a control well, adding 2.5 mu L of the ASPE reaction product obtained in the step (3), sucking and beating uniformly, and incubating for 30min at 37 ℃ under the hybridization reaction condition of denaturation at 96 ℃ for 90 s;

(5) and (4) detecting a result: the hybridized microspheres were resuspended in 100. mu.L of 1 × hybridization buffer containing 6.5. mu.g/mL streptavidin-phycoerythrin, incubated at 37 ℃ for 20min, and then detected on a Luminex 200 instrument.

In a preferred embodiment of the present invention, in step (1), the multiplex PCR reaction system is: total volume 50. mu.l, gDNA 50ng, 2. mu.l, 10 XPCR reaction buffer 5. mu.l, Taq^TM0.25 μ l of Hot Start enzyme, 2.5mM of each dNTP, 4 μ l of each dNTP, 10 μ M of each primer, 0.5 μ l of each primer, and 30.75 μ l of deionized water;

the procedure for the PCR amplification reaction was: pre-denaturation: 94 ℃ for 30s, cycle: 94 ℃ 30s, annealing 57 ℃ 30s, extension 72 ℃ 30s, a total of 5 times, 94 ℃ 30s, annealing 55 ℃ 30s, extension 72 ℃ 30s, a total of 30 times, final extension: 10min at 72 ℃;

in step (3), the ASPE reaction system is: after purification, 2. mu.l of PCR product, 5. mu.l of 10 XPCR reaction buffer, 0.35. mu.l of 400. mu.M Biotin-dCTP, 100. mu.M each of dATP, dGTP and dTTP, 1. mu.l total of 100mM dCTP, 0.3. mu.l of Taq^TM0.1 mul of Hot Start enzyme, 500nM of each of the TAG-ASPE primers, 1 mul in total, and 13.55 mul of deionized water;

the procedure for the ASPE extension reaction was: pre-denaturation: 90s at 94 ℃; and (3) circulation: 30s at 94 ℃, 30s at 57 ℃ and 1min at 74 ℃ for 40 times.

The primer and the kit provided by the invention can simultaneously detect related genes of various common cardiovascular disease drugs by using a liquid chip technology, and have the following beneficial technical effects:

1. the liquid-phase chip detection kit comprises 15 pairs of specific primers, is combined with MagPlex microspheres to simultaneously amplify and detect the polymorphism of 15 common cardiovascular drug-related target genes CYP2C9, VKORC1, CYP4F2, CYP2C19, PON1, CYP2D6, ADRB1, ALDH2, GP1BA, PEAR1 and AGTR1 in one-step PCR reaction, has high detection flux and high cost performance, can provide medication guidance for patients with cardiovascular diseases through one-time detection, provides laboratory basis for clinical rational medication, and has great clinical application value.

2. The kit has very good specificity, and no cross reaction exists between the designed primer and probe sequences.

3. The kit has very good sensitivity and stability in actual detection.

4. The kit provided by the invention has simple operation steps, 15 mutation sites can be simultaneously detected by PCR combined with ASPE reaction, and sample pollution possibly caused in repeated PCR operation processes is avoided, so that the detection accuracy is greatly improved.

5. The detection result is well matched with the sequencing result, the type of the target gene polymorphism sites related to cardiovascular drugs can be accurately detected, the result is stable and reliable, and compared with the conventional SNP detection method, the detection cost is greatly reduced.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIGS. 1A-1D in FIG. 1 show the principle of allele-specific primer extension (ASPE) in liquid-phase chip technology.

Detailed Description

The inventor of the invention develops a primer group, a kit and a kit using method capable of simultaneously detecting related gene polymorphism of a plurality of cardiovascular drugs for the first time through extensive and intensive research.

Term(s) for

Liquid phase chip

The term "liquid phase chip" as used herein is a biochip with a completely new concept. The core of the technology is that tiny polystyrene beads (5.6 mu m) are coded by a fluorescent staining method, and then microspheres (or called fluorescent coding microspheres) of each color are covalently linked with probes, antigens or antibodies aiming at specific detection objects. When the method is used, the coded microspheres for different detection objects are mixed, then a trace of sample to be detected is added, the target molecules in suspension and the molecules crosslinked on the surfaces of the microspheres are specifically combined, and up to 100 different biological reactions can be simultaneously completed in one reaction hole. Finally using Luminex^TMAnd (4) analyzing by analysis software, and respectively identifying the fluorescence intensity of the coded microspheres and the fluorescence intensity of the reporter molecules on the detection microspheres by an instrument through two beams of laser. Because the molecular hybridization or immune reaction is carried out in the suspension solution, the detection speed is extremely high, and up to 100 indexes can be simultaneously detected in a trace liquid reaction system. See figure 1 for experimental principles.

Allele Specific Primer Extension (ASPE)

The term "Allele Specific Primer Extension (ASPE)" as used herein is a solution-based sequence-Specific enzymatic reaction technique that can be used to determine multiple SNPs in a single tube. The ASPE method involves two stages, first an enzymatic reaction that determines the target genotype, and then capture on the surface of solid microspheres for detection. This technique allows the detection of new templates (i.e., ASPE amplification products of the invention) using sequence-labeled microspheres (i.e., microspheres coupled to addressing probe sequences of the invention) using a solution phase kinetics phase. This is done with the aid of an appropriate capture sequence linked to an allele-specific oligonucleotide.

The template for the ASPE reaction is the amplified PCR fragment. In designing the extension primers, the SNP should be present at the 3 'end of the allele-specific primer for accurate hybridization and the capture sequence (TAG sequence) should be located at the 5' end of the primer. In the allele-specific primer extension step, a polymerase extends the primer by incorporating Biotin-labeled dNTPs (Biotin-dCTP). Extension occurs only when the 3' end of the allele-specific primer binds to the homologous allele sequence, and thus, the extension product comprises the TAG sequence + the target gene locus + an amplified fragment comprising the biotin label.

The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for the purpose of illustration only and are not intended as a definition of the limits of the invention. The experimental procedures, for which specific conditions are not indicated in the following examples, were carried out according to conventional experimental conditions, such as those described in Sambrook et al, the molecular cloning instructions (fourth edition) or according to conditions recommended by the manufacturer of the product.

Examples

The reagents and apparatus used in the present invention were as follows:

the DNA extraction Kit TIANAmp Genomic DNA Kit is purchased from Beijing Tiangen Biotechnology, Inc.; the multiplex PCR kit TaqTM Hot Start Version was purchased from TaKaRa; platinum^TMGenoType Tsp DNA polymerase, exonuclease I-shrimp alkaline phosphatase (ExoSAP-IT), Biotin-dCTP, dNTP, streptavidin-phycoerythrin (SA-PE), etc. were purchased from Saimer Feishal science; the LifeECO gene amplification instrument is purchased from China perspective Life technologies, Inc.; BioDrop protein nucleic acid analyzers were purchased from haworth biotechnology ltd; MagPlex-Tag microspheres and Luminex 200 were purchased from Luminex corporation, USA.

Example 1 primer set for detecting cardiovascular drug-related genes by liquid chip technology

Design of primers and probes:

base sequences around 15 mutation points are searched in Genbank according to the rs number of SNP disclosed on NCBI, and specific primers for amplifying the following target gene sites related to common cardiovascular drugs are designed by using Primer 6.0 Primer design software: the polymorphism rs1799853 of C430T polymorphism of CYP2C9 gene, the polymorphism rs1057910 of A1075C polymorphism of CYP2C9 gene, the polymorphism rs9923231 of G1639A polymorphism of VKORC1 gene, the polymorphism rs 1347T polymorphism rs2108622 of C1344F 2 gene, the polymorphism rs4244285 of G681A polymorphism of CYP2C19 gene, the polymorphism rs4986893 of G636A polymorphism of CYP2C19 gene, the polymorphism rs12248560 of C806 polymorphism of CYP2C19 gene, the polymorphism rs662 of Q192 19 of PON 19 gene, the polymorphism rs16947 of C2850 19 gene, the polymorphism rs 19 of CYP2D 19 gene, the polymorphism rs 6715 rs1801253 of ADRB 19 gene, the polymorphism G36504 polymorphism of ALDH 19 gene, the polymorphism T5 rs 19 of GP1 19 gene, the polymorphism rs 224224512772 gene, the polymorphism of AR 11651513672 gene and the polymorphism of AG513672 gene AG513672.

Specific primers aiming at the site polymorphism of 15 target genes are designed according to the nucleotide sequence of the site polymorphism of the genes, are used for amplifying DNA fragments of the 15 target gene sites, and ASPE primer sequences are respectively designed aiming at wild types and mutant types of the 15 target gene sites, and the total number is 30. The ASPE primer consists of a Tag sequence and a specific primer sequence. In the process of designing the primers, in order to avoid cross reaction between the target genes for joint detection, firstly, non-specific amplification is avoided between fifteen pairs of specific primers, so that the Tm difference value of the PCR forward primer and the reverse primer is less than 5 ℃, and meanwhile, the annealing temperature of the first 5 PCR cycles is increased by 2 ℃ and is set as 57 ℃; secondly, cross extension between ASPE primers is avoided, when a specificity experiment is carried out, if cross reaction occurs between the ASPE primers, the ASPE primers are redesigned, the highly specific ASPE primers are designed by using PrimerPlex software, mutation points are positioned at the 3' end of the designed primers, and PCR amplification products are coupled with the ASPE capture sequence primers and extended through an ASPE reaction; in addition, a Tag sequence is designed according to the designed ASPE-specific primer fragment so as to minimize the possible secondary structure formed between the Tag sequence and the ASPE-specific primer fragment.

Because a plurality of primers and templates in the multiplex PCR system exist in the same reaction tube, mutual interference is easily caused in the experimental process, and dimers are easily formed among the primers, thereby influencing the extension of the ASPE primers and even causing the failure of the experiment. Therefore, the primer design is the key of the invention and is also the basis for Luminex liquid phase chip detection.

The inventor of the invention overcomes the difficulty of the design of the multiplex PCR primer, and needs BLAST search to eliminate the homologous region before the primer is designed, and ensures that the PCR amplification product between the primers is at 100-700 bp; the eight pairs of primers are concentrated into 1 multiplex PCR system for PCR amplification; if the amplification efficiency of part of the sites is not enough, the concentration or design of the PCR primers is adjusted, or the primer combination of the multiplex PCR is adjusted until all the sites can be amplified in a relatively balanced manner, and the optimization of the PCR is completed. After a large number of experiments, the designed specific amplification primers and the ASPE extension primers are screened and improved, and finally, 15 pairs of specific amplification primers (see Table 1) for amplifying 15 target gene templates and 30 ASPE extension primers (see Table 2) for extending target gene loci are preferably selected.

TABLE 1 specific amplification primers for amplification of target sequences comprising target gene loci

The specific amplification primers for amplifying the target sequence containing the target gene locus are synthesized by Shanghai biological engineering technology service, Inc., and each synthesized primer is respectively prepared into 10 mu M stock solution by using deionized water.

TABLE 2.15 target Gene ASPE extension primer sequences

Note: and the bold font is a detection site.

The ASPE extension primers are synthesized by Shanghai biological engineering technology service company Limited, and each synthesized primer is prepared into 500nmol/L stock solution by using deionized water.

Example 2. kit for simultaneously detecting polymorphisms of a plurality of cardiovascular drug-related target genes using a liquid chip technique of the present invention the kit of the present invention comprises:

1. primers in tables 1 and 2 were designed in example 1.

2.30 MagPlex-Tag microspheres conjugated with addressing probe sequences capable of complementary pairing with Tag sequences in the ASPE extension primers. The 30 microsphere numbers and the anti-Tag sequences coupled on the microspheres are shown in Table 3:

TABLE 3.30 MagPlex-Tag microspheres coupled with addressing Probe sequences (anti-Tag sequences)

30 selected MagPlex-Tag microspheres were purchased from Luminex, USA at a concentration of 2.5X 10⁶And each microsphere is used by diluting to 2500 microspheres, wherein the addressing probe sequence is coupled on the microsphere and is complementarily matched with the Tag sequence in the ASPE primer.

3. Streptavidin-phycoerythrin hybridization buffer solution, PCR reaction solution, DNA polymerase and negative control; wherein the streptavidin-phycoerythrin hybridization buffer solution is purchased from Saimer Feishell science and technology company, the concentration is 6.5 mug/mL, the multiplex PCR reaction solution is 10xbuffer, and the streptavidin-phycoerythrin hybridization buffer solution and DNA polymerase are from Taq^TMThe HotStartVersion kit was purchased from TaKaRa, and the negative control was microspheres of the same amount plus deionized water.

Example 3 detection of common cardiovascular drug-related target Gene polymorphisms Using the primer set and kit of the invention

This example used the primer set of example 1 and the kit of example 2 to detect 15 target gene polymorphisms of common cardiovascular drugs.

The specific experimental steps are as follows:

1. multiplex PCR amplification reaction: a total of 14 whole blood samples (blood samples are from Dongguan Hospital) are extracted, and according to the experimental requirements, another negative control is arranged to extract DNA. A multiplex PCR reaction (50. mu.l) was prepared using the specific amplification primers in Table 1 of example 1:

composition (I)	Dosage (mu l)
		gDNA	50ng(2μl)
10 XPCR reaction buffer	5
		Taq^TM HotStar	0.25
dNTPs (2.5 mM each)	4
		Upstream and downstream primers (10. mu.M)	0.5 each
Deionized water	30.75

The PCR amplification parameters were designed as follows:

pre-denaturation: 94 ℃ for 30s

And (3) circulation: 30s at 94 ℃, 30s at 57 ℃ for annealing, 30s at 72 ℃ for extension, 5 times in total

30s at 94 ℃, 30s at 55 ℃ for annealing, 30s at 72 ℃ for extension, 30 times in total

Final extension: 72 ℃ for 10min

In order to verify whether a PCR product obtained by carrying out PCR reaction by using the designed primer is a target sequence containing a target gene locus, a part of the PCR product is reserved and sent to Guangzhou Egyptian limited organism for sequencing, and the sequencing result has good goodness of fit with the target PCR product.

Purification of PCR products: after the PCR reaction is finished, redundant dNTP, primers and single-stranded products remain in the products, the subsequent ASPE extension reaction is seriously influenced, and the impurities can be removed by using ExoSAP-IT. 2.5 ul ExoSAP-IT +5 ul PCR product 37 ℃ temperature in 30min, to degrade excess primer, single-stranded DNA, dNTP, and then 80 ℃ temperature in 20min to enzyme inactivation, purification of PCR products.

ASPE reaction: purified PCR product is Taq in ASPE reaction system^TMThe extension reaction is continued only when the first base at the 3' end of the primer is complementary to the detection site of the amplified target sequence by the action of HotStar polymerase, and if not, the extension reaction is terminated. The ASPE reaction is carried out according to the following optimized reaction system:

TABLE 4 ASPE optimized reaction System

The ASPE reaction parameters were designed as follows:

pre-denaturation: 90s at 94 ℃;

and (3) circulation: 30s at 94 ℃, 30s at 57 ℃, 1min at 74 ℃ for 40 times;

the optimized detection system shows that the primer designed by the invention has strong specificity and no cross reaction. The ratio of the Biotin-labeled dCTP (Biotin-dCTP) concentration to unlabeled dCTP concentration in the ASPE reaction system is maintained at 3: 1, which is one of important factors for successful detection system, and when the ratio is too high, the ASPE primer is effectively extended, and when the ratio is too low, the fluorescence signal is weak.

4. And (3) hybridization reaction: selectingSelected 30 MagPlex-Tag microspheres were purchased from Luminex, USA at a concentration of 2.5X 10⁶Each/ml, diluted to 100 per microliter, 25 μ L of diluted microspheres (containing 2500 each microspheres) were loaded onto a 96-well hybridization plate, and the same amount of microspheres and deionized water were added to the control wells, 2.5 μ L of ASPE reaction product was added, and the blots were blotted well. The whole operation process is protected from light. Hybridization conditions: denaturation at 96 ℃ for 90s and incubation at 37 ℃ for 30 min.

5. And (4) detecting a result: the hybridized microspheres were resuspended in 100. mu.L of 1 × hybridization buffer containing 6.5. mu.g/mL streptavidin-phycoerythrin, incubated at 37 ℃ for 20min and detected on a Luminex 200 instrument.

Thirdly, analyzing the detection result and data

The results of the detection by the Luminex analyzer are shown in Table 5.

The requirements of the instrument for fluorescence value (MFI) are: the detection site MFI ratio is detection site MFI/(mutant MFI + wild MFI), the MFI ratio is homozygote when the ratio is more than 0.75 or less than 0.25, and heterozygote when the ratio is between 0.25 and 0.75. The detection result obtained by using the kit has higher resolution, the MFI ratio of homozygote is basically more than 0.9 or less than 0.1, and heterozygote is between 0.35 and 0.65, which indicates that the resolution of the detection result is obviously superior to the genotyping standard provided by Luminex company.

The results of the sequencing method detection and the liquid phase chip are compared, and the coincidence rate of the detection results of the typing method provided by the invention is calculated. The coincidence rate of the detection result of 30 genotypes of cardiovascular medicine related target gene loci of 14 whole blood samples detected by the method and the sequencing result reaches 100%. Therefore, the liquid chip detection primer group and the kit provided by the invention can accurately detect the type of the target gene polymorphism sites related to cardiovascular drugs, and the result is stable and reliable.

Example 4 detection specificity analysis and clinical specimen verification experiment of primers and kit of the invention

In order to ensure that the designed specific ASPE primer of the invention can only carry out extension reaction at the corresponding detection site, the specific detection is carried out aiming at single-site mutation in the embodiment, the result shows that the ASPE primer probe has no cross reaction with other mutation sites, the result of Luminex 200 detecting 15 target genes of 13 samples is shown in Table 6, and the carrying frequency of each allele is basically consistent with that of Hatmap. All detection samples are sent to Guangzhou Eji biological Limited for sequencing, and the Luminex detection result is completely consistent with the sequencing result, which shows that the ASPE primers of the primers and the kit have high specificity.

Example 5 assay of detection sensitivity of primer set and kit of the invention

The diluted sample was tested according to the optimal reaction system of example 3, with DNA concentration between 100-0.75 ng. As the DNA concentration decreased, the MFI values tested decreased gradually, and the difference in MFI ratios between all alleles tested at different DNA concentrations was less than 0.03, the test results are shown in Table 7. The minimum concentration of the genotype of the target site can be clearly distinguished in the three detections is 1.5ng, which indicates that the primer group and the kit have high detection sensitivity.

So far, a technical platform capable of simultaneously detecting common 15 target gene loci of cardiovascular drugs is not found, the primer group and the kit disclosed by the invention have the advantages of high detection flux, high cost performance, great clinical application value, capability of simultaneously detecting gene polymorphisms of drugs such as warfarin, clopidogrel, nitroglycerin, aspirin and beta receptor blocker, capability of providing timely and accurate medication information for clinic, low cost, high specificity, high sensitivity, simplicity in operation, flexibility in combination and the like.

Sequence listing

<110> Dongguan city Hospital in Thick street

<120> specific primer and reagent for detecting cardiovascular disease drug gene polymorphism by liquid phase chip technology

Box

<130> L019PAF20180307

<160> 90

<170> PatentIn version 3.5

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ttcttcatta acttctaatc ttacaagagg agcattgagg acc 43

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tacttcttta ctacaattta caacggtggg gagaaggtca at 42

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ctttctcata ctttcaacta atttggtggg gagaaggt 38

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ttaaacaatc tactattcaa tcactgaaaa acaaccattg gccg 44

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taacttacac ttaactatca tctttgaaaa acaaccattg gcca 44

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caataaacat tctttacatt ctcactcagg gtccggccac at 42

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ggttgttgat gtccatcg 18

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ctttcttaat acattacaac atacagtaat ttgttatggg ttccc 45

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tcaaactctc aattcttact taatagtaat ttgttatggg ttcct 45

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acaaatatct aactactatc acaaattgta agcaccccct gg 42

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ctttatcaaa ttctaattct caacattgta agcaccccct ga 42

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acactcattt aacactattt cattgtgtct tctgttctca aagc 44

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acacttatct ttcaattcaa ttacgtgtct tctgttctca aagt 44

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gaatagacag tgaggaatgc 20

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tacattcaac actcttaaat caaaattttc ttgaccccta cttaca 46

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atactttaca aacaaataac acacattttc ttgaccccta cttacg 46

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tactacttct ataactcact taaagcttca atgatgagaa cctgg 45

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actacttatt ctcaaactct aatagcttca atgatgagaa cctga 45

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ttggtagtga ggcaggtat 19

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caaatacata atcttacatt cactggctgc aggcatacac ta 42

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cataatcaat ttcaactttc tactgcttgc attattgcag gaacc 45

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aataacaact cactatatca taacgcttgc attattgcag gaacc 45

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Claims

1. A primer group for simultaneously detecting 15 cardiovascular drug related target gene polymorphisms by using a liquid chip technology comprises 15 groups of primers, wherein each group of primers comprises a pair of specific amplification primers for detecting one target gene polymorphism and two ASPE primers respectively aiming at a wild type and a mutant type of the target gene;

the detected target gene polymorphisms were: CYP2C9-C430T rs1799853, CYP2C9-A1075C rs1057910, VKORC1-G1639A rs9923231, CYP4F2-C1347T rs2108622, CYP2C19-G681A rs4244285, CYP2C19-G636 rs4986893, CYP2C19-C806 19 rs 48560, PON 19-Q192 19 rs662, CYP2D 19-C2850 19 rs 947, CYP2D 19-C100 19 rs1065852, ADRB 19-G1165 rs1801253, ALDH 19-G19 rs671, GP1 19-T5 19 rs 22422422422493, PEAR 72G > A27759 and AGTR 19-511165186A 5172;

the sequences of the primer sets for detecting the polymorphism of the target gene are as follows:

specific amplification primers for detecting CYP2C19-G681A rs4244285 are shown as sequences SEQ ID NO.17 and SEQ ID NO.18, and ASPE primers are shown as sequences SEQ ID NO.19 and SEQ ID NO. 20;

2. The primer set according to claim 1, wherein the ASPE primer comprises two parts of a Tag sequence at 5 ' end and a specific primer sequence for target gene polymorphism at 3 ' end, and the first base at 3 ' end of the ASPE primer is complementary-paired with the target gene to be detected.

3. The primer set according to claim 2, wherein the specific primer sequence included in the ASPE primer is represented by the sequence SEQ ID No. 61-90.

4. Use of the primer set according to any one of claims 1 to 3 in the preparation of a kit for simultaneously detecting 15 cardiovascular drug-related target gene polymorphisms by using a liquid chip technology.

5. A kit for simultaneously detecting the polymorphism of a plurality of cardiovascular drug-related target genes by using a liquid chip technology, the kit comprising the primer set of claim 1 and 30 microspheres, wherein,

6. The kit of claim 5, further comprising streptavidin-phycoerythrin hybridization buffer, PCR reaction solution, DNA polymerase and negative control.

7. The kit of claim 5, wherein the sequence of the specific primer at the 3' end included in the ASPE primer is shown as the sequence SEQ ID NO. 61-90.

8. The kit of claim 6, wherein the negative control is distilled water plus microspheres.