CN112301121A - Probe, primer and kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene - Google Patents

Abstract

The invention discloses a probe, a primer and a kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene, wherein the sequence of the probe is shown as SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 7, or both ends of the sequence of the SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 7 are modified by groups. The probe, the primer and the kit for detecting the polymorphism of the CYP2C9 gene and the VKORC1 gene have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like when the polymorphism of the CYP2C9 gene and the VKORC1 gene is detected.

Description

Probe, primer and kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene

Technical Field

The invention relates to the field of molecular biology, in particular to a probe, a primer and a kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene.

Background

Warfarin is the earliest, the most and the most widely used oral anticoagulant drugs in clinic at present, and has been applied to anticoagulant therapy of various diseases, such as primary and secondary prevention of venous thromboembolic diseases (VTE), prevention of atrial fibrillation thromboembolism, valvulopathy, artificial valve replacement, thrombosis in heart cavities and the like, but the clinical curative effect and adverse reaction of warfarin are greatly different from individual to individual, the dosage is difficult to master, and particularly, serious bleeding complications are easily caused at the early stage of warfarin anticoagulant therapy. It is estimated that 15.2% of people develop blood side effects each year, with fatal major bleeding accounting for 3.5%, and warfarin-stabilizing doses varying by more than 20-fold among individuals.

CYP2C9 is an important member of the second subfamily of cytochrome P450 enzymes (CYP) and accounts for 20% of the total amount of liver microsomal P450 protein. CYP2C9 participates in the hydroxylation metabolism of various drugs such as anticoagulants, anticonvulsants, hypoglycemic drugs, nonsteroidal antipyretic analgesic and anti-inflammatory drugs, antihypertensive drugs and diuretic drugs, wherein warfarin, tolbutamide and phenytoin are all drugs with narrow therapeutic indexes. Changes in CYP2C9 activity can lead to large changes in the in vivo concentration of these drugs and even to the occurrence of serious adverse drug reactions. CYP2C9 x 2(rs1799853, C430T, Arg144Cys) and CYP2C9 x 3(rs1057910, A1075C, Ile359Leu) both cause the activity of CYP2C9 to be reduced, and the activity of CYP2C9 x 3 homozygote individual enzyme is only 4-6% of that of wild-type homozygote genotype individuals (carrying CYP2C9 x 1 or Arg144/Ile359 alleles) at the site. CYP2C9 genetic polymorphism causes variation of enzyme activity, thereby causing phenomena of drug metabolism ethnicity and individual difference.

Vitamin k oxidoreductase is the target of the anticoagulant drug warfarin. Genetic variation of the gene VKORC1 encoding vitamin K epoxide reductase complex 1 can affect warfarin sensitivity by affecting VKORC1 expression. The single nucleotide mutation rs9923231 located in the promoter region (-1639G > A) of the gene can affect the expression of VKORC1, and is one of the main reasons for individual difference of warfarin dosage. Compared with the AA genotype patients at the site, the average warfarin dose of-1639 GA and GG genotype patients is increased by 52% (95% CI: 41-64%) and 102% (95% CI: 85-118%), respectively. The specific gravity of the warfarin dose effect of the VKORC1 polymorphism varied from race to race, and the effects of the-1639 GA and GG genotypes on white warfarin dose were 10% and 50% higher than those of Asians, respectively. Overall, the VKORC1 polymorphism may account for individual differences in warfarin dosage of about 27% among different ethnic groups of people. The allele frequencies of VKORC1-1639A alleles in Asian, Caucasian and Caucasian populations are 91.17%, 38.79% and 10.81%, respectively (according to the results of the thousand-people database: 92%, 40% and 7% in Asian, Caucasian and Caucasian populations), and the ethnic difference in frequency distribution has a good correlation with warfarin dosage difference. The VKORC1 polymorphism also affects the clinical outcome of warfarin administration.

A large number of researches show that the site polymorphism of warfarin metabolic enzyme genes CYP2C9 and CYP2C9 and 3 and the site polymorphism of warfarin target gene VKORC1-1639G > A in China are closely related to the anticoagulation curative effect, and the warfarin dosage difference required by different genotype patients is obvious. The FDA in the united states updated the instructions for warfarin in 2010 by suggesting that the initial dose of warfarin be considered in combination with VKORC1 and CYP2C9 genotypes to achieve the goal of aiding in optimizing warfarin use and reducing the risk of bleeding.

The mutation detection method commonly applied at present is a direct DNA sequencing method, and the PCR product is directly subjected to DNA sequence analysis, so that mutation sites can be defined, but the defects of time and labor waste, high cost, inapplicability to detection of a large number of samples and the like exist. Therefore, it is necessary to develop a method suitable for the detection of large-scale samples.

Disclosure of Invention

The invention aims to provide 3 groups of probes for detecting polymorphism of CYP2C9 gene and VKORC1 gene, wherein the sequence of the probes is shown as SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 7; or the two ends of the sequences of SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 7 are modified by groups;

wherein the modifying group is: the 5' modifying group is a fluorescent group, such as FAM, HEX, CY 5; the modifying group of 3' is a quenching group, such as BHQ1 and BHQ 2;

specifically, the probe for detecting the polymorphism of the CYP2C9 × 2(rs1799853, C430T) gene provided by the invention is as follows: 1HEX-CGAGCATTGAGGAC SEQ ID NONGTGTTCAAGAGGCTCG-BHQ1；

Wherein N is a variant base, specifically C > T;

the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-26 bp (5' -ATTGAGGAC)NGTGTTCAAGAG-3 '), which is flanked by two reverse complementary stem sequences, the stem sequence being (5' -CGAGC … … GCTCG-3).

The probe for detecting CYP2C9 x 3(rs1057910, A1075C) gene polymorphism provided by the invention is as follows: SEQ ID No. 4FAM-CCAGAGATACNTTGACCTTCTGG-BHQ1；

Wherein N is a variant base, specifically A > C;

the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-18bp (5' -GATAC)NTTGACCT-3 ') flanked by two reverse complementary stem sequences (5' -CCAGA … … TCTGG-3).

The kit is used for detecting VKORC1 (-1639G)>A) The probe of the (rs9923231) gene polymorphism is as follows: 7CY5-CCACCGCACC SEQ ID NONGGCCAATGGTGG-BHQ2；

Wherein N is a variant base, specifically G > A;

the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-18bp (5' -GCACC)NGGCCAAT-3 '), flanked by two reverse complementary stem sequences (5' -CCACC … … GGTGG-3).

The design principle of the probe is as follows: probes and primers are respectively designed according to CYP2C9 gene polymorphism rs1799853, rs1057910 and VKORC1 gene polymorphism rs9923231, and the probes are required to be in a stem-loop state when a DNA template does not exist at an annealing temperature. The quenching group adopted by the probe is BHQ1 or BHQ2, the quenching space range is very small, and the fluorescence can be well quenched only when the molecular beacon is in a stem-loop structure by matching with a fluorescent group with short emission wavelength, such as HEX. When the loop sequence is complementary with the target DNA sequence, the optimal PCR primer is determined to be 40-45bp in size, and the length of the PCR product is 100-150 bp.

The invention also provides 3 groups of primers for detecting the polymorphism of the CYP2C9 gene and the VKORC1 gene, wherein the sequences of the primers are shown as SEQ ID NO. 2, 5 and 8 (primer 1) and SEQ ID NO. 3, 6 and 9 (primer 2):

CYP2C9*2(rs1799853，C430T)

SEQ ID NO.:2 5′-AGGTGACACTATAGAATATCAGCTTCCTCTTTCTTGCC-3′

SEQ ID NO.:3 5′-GCAAGCCCTCACGTAGCGAACCACCCTTGGTTTTTCTCAA-3′。

CYP2C9*3(rs1057910，A1075C)

SEQ ID NO.:5 5′-AGGTGACACTATAGAATAGAACGTGTGATTGGCAGAAA-3′

SEQ ID NO.:6 5′-GCAAGCCCTCACGTAGCGAATGTCACAGGTCACTGCATGG-3′。

VKORC1(-1639G>A)(rs9923231)

SEQ ID NO.:8 5′-AGGTGACACTATAGAATACTCCTGACCTCAAGTGATCCA-3′

SEQ ID NO.:9 5′-GCAAGCCCTCACGTAGCGAACCTCTGGGAAGTCAAGCAAG-3′。

the invention also provides a kit for detecting the polymorphism of the CYP2C9 gene and the VKORC1 gene, which comprises the probe and/or the primer;

the kit further comprises Taq enzyme, dNTP, and/or Mg²⁺And instructions for use.

The invention also provides a method for detecting the polymorphism of the CYP2C9 gene and the VKORC1 gene, which comprises the following steps:

(1) collecting a sample and extracting DNA;

(2) carrying out fluorescent quantitative PCR reaction by using the probe and the primer;

(3) and (3) analyzing the result by using a PCR instrument matched software, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks.

Wherein the total PCR reaction system is 15ul (comprising 7.5ul of PCR Mix, 0.5ul of each forward primer solution in three groups, 0.5ul of each reverse primer solution in three groups, 0.1ul of each probe in 3 groups, 2ul of sample DNA and 2.2ul of sterilized double distilled water); carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 92-97 ℃ for 5-15 minutes; denaturation at 92-97 deg.C for 10-30 s, annealing at 57-65 deg.C for 10-30 s, extension at 70-75 deg.C for 10-30 s, and 40-50 cycles; extension at 72 ℃ for 10 min; denaturation at 92-97 deg.C for 1 min, renaturation at 40 deg.C for 1 min, and real-time monitoring of fluorescence signal at 45-80 deg.C, and recording 5 times at 1 deg.C per time.

The invention detects the polymorphism of CYP2C 9X 2, CYP2C 9X 3 and VKORC1 genes, can realize the rapid screening of the polymorphism of CYP2C 9X 2, CYP2C 9X 3 and VKORC1 genes, is beneficial to establishing and evaluating the dosage of individual warfarin, thereby achieving the purpose of assisting the optimization of the warfarin and reducing the bleeding risk.

The invention utilizes a fluorescent probe capable of specifically identifying a nucleic acid sequence, releases fluorescent dye through conformational change after hybridization with a target sequence, and judges a typing result according to peak patterns at different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected. Compared with other genetic typing techniques, the method is simple to operate, 96 cases of detection can be completed within 2-3 hours, the method has the advantages of high sensitivity, good specificity, rapidness, high-flux detection and the like, the fluorescence labeling probe can use different fluorophores at different sites, multiple detections can be realized, the detection result can be obtained by directly detecting the fluorescence signal in the PCR process, the genotyping is clear, the PCR post-treatment or the electrophoresis detection is not needed, and the real closed-tube operation can be realized.

Drawings

FIG. 1 is a schematic view of a probe stem-loop structure

FIG. 2, three peak types (CC type, TT type, CT type) at rs1799853 locus

Three peak types (CC type, AA type, AC type) at the locus rs1057910 in FIG. 3

FIG. 4, three peak types (GG type, AA type, AG type) at the rs9923231 site

FIG. 5 is a graph showing melting curves of multiple samples

Detailed Description

Example 1: probe and primer design

The invention designs probe and primer sequence aiming at CYP2C9 x 2C T, CYP2C9 x 3A C, VKORC1-1639G > A SNP locus. The specific principle is that the fluorescent probe and a target sequence are hybridized to release fluorescent dye through conformational change, and a genotyping result is judged according to peak graphs and Tm values of different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected.

And designing a probe and a primer to realize that the probe is in a stem-loop state when the template does not exist at the annealing temperature. CYP2C9 x 2 is shown in FIG. 1, and comprises a loop sequence and a stem sequence which is reversely complementary on two sides of the loop sequence, wherein the total length is 31bp, and the loop sequence is 21bp (5' -ATTGAGGAC)NGTGTTCAAGAG-3'), the two ends of which are respectively provided with 5 basic groups to form a stem sequence; and the stem sequences at the two ends are just complementary; the adopted quenching group is BHQ1 matched with a fluorescent group HEX with short emission wavelength. The loop sequence is complementary with the target DNA sequence, the size of the PCR primer is determined to be 38-40bp, and the length of the PCR product is 139 bp.

CYP2C9 x 2(rs1799853, C430T) primers and probe sequences were as follows:

sequence of primer 1: 5'-AGGTGACACTATAGAATATCAGCTTCCTCTTTCTTGCC-3', respectively;

sequence of primer 2: 5'-GCAAGCCCTCACGTAGCGAA CCACCCTTGGTTTTTCTCAA-3', respectively;

sequence of the probe: 5' -HEX-CGAGCATTGAGGACNGTGTTCAAGAGGCTCG-3′BHQ1。

Wherein N is a variant base, specifically C > T;

CYP2C9 x 3(rs1057910, a1075C) primer and probe sequences were as follows:

sequence of primer 1: 5'-AGGTGACACTATAGAATAGAACGTGTGATTGGCAGAAA-3', respectively;

sequence of primer 2: 5'-GCAAGCCCTCACGTAGCGAATGTCACAGGTCACTGCATGG-3', respectively;

sequence of the probe: 5' -FAM-CCAGAGATACNTTGACCTTCTGG-3′BHQ1。

Wherein N is a variant base, specifically A > C

VKORC1(-1639G > A) (rs9923231) primers and probe sequences were as follows:

sequence of primer 1: 5'-AGGTGACACTATAGAATACTCCTGACCTCAAGTGATCCA-3'

Sequence of primer 2: 5'-GCAAGCCCTCACGTAGCGAACCTCTGGGAAGTCAAGCAAG-3', respectively;

sequence of the probe: 5'-CY5-CCACCGCACCNGGCCAATGGTGG-3′BHQ2。

Wherein N is a variant base, specifically G > A.

The above-mentioned probe and primer were synthesized by Biotechnology engineering (Shanghai) Ltd.

Example 2: standard substance for detecting different genotypes

1. Plasmids CYP2C9 x 2, CYP2C9 x 3 and VKORC1 are used for constructing and preparing wild type standard plasmids and mutant type standard plasmids (plasmid sources and the synthesis of plasmids containing target genes are synthesized by a company Limited in Biotechnology (Shanghai) and biological engineering (Shanghai) which contain target genes rs1799853, rs1057910 and rs9923231 sites, the sequence accuracy is determined by sanger sequencing, the wild type standard plasmids rs1799853 genotype is CC, the rs1057910 genotype is AA, the rs9923231 genotype is GG, the mutant type standard plasmids rs1799853 genotype is TT, the rs1057910 genotype is CC and the rs9923231 genotype is AA, and the DNA concentration of the standard plasmids is normalized to 10 ng/ul.

2. The probe and primer in example 1 were used.

3. And (3) PCR reaction system:

1) sequentially adding 7.5ul of PCR Mix, 0.5uM of 3 groups of forward primer solutions, 0.5uM of 3 groups of reverse primer solutions and 0.1uM of 3 groups of probes into each PCR reaction hole, then respectively adding 2ul of wild type standard plasmid DNA, mutant type standard plasmid DNA and mixed type DNA (the wild type standard plasmid and the mutant type standard plasmid are mixed according to a ratio of 1: 1) into 3 different PCR reaction holes, and supplementing 15ul of sterile redistilled water;

2) carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.

4. And (3) analyzing results by using a PCR instrument matched software SLAN, and displaying different genotypes based on the difference of Tm values displayed by hybridization peaks formed by different channels. The peak at rs1799853 locus appeared at 63 ℃ and was CC genotype, and at 57 ℃ it was TT genotype, and at both positions, the peak was CT genotype (FIG. 2). The peak at rs1057910 locus appeared at 54 ℃ as AA genotype, 60 ℃ as CC genotype, and both positions had peaks as AC genotype (FIG. 3). The peak at rs9923231 locus appeared at 68 ℃ and was the GG genotype, the AA genotype at 63 ℃, and the peak at both positions was the AG genotype (FIG. 4).

Example 3: double-blind experimental investigation using warfarin personalized medicine related genes (CYP2C9 x 2, CYP2C9 x 3 and VKORC1) detection kit

1. Extracting the genome DNA of oral epithelial cells of 50 healthy volunteers in Shanghai region by a silica gel adsorption method, detecting the concentration and purity of the DNA by an electrophoresis gel imaging method, and marking the concentration of the DNA of a sample to be detected to 10 ng/ul.

2. The detection method comprises the following steps: sequentially adding 7.5ul of PCR Mix, 0.5uM of forward primer solution, 0.5uM of reverse primer solution and 0.1uM of probe into each PCR reaction hole, simultaneously detecting a weak positive control (the rs1057910 genotype is AC and the rs9923231 genotype is AG), a negative control (the rs1057910 genotype is AA, the rs1799853 genotype is CC and the rs9923231 genotype is GG) and a sample to be detected, adding DNA2ul into each reaction hole, and supplementing 15ul of sterilized redistilled water; carrying out reaction on a fluorescent quantitative PCR detector, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.

3. And analyzing the result by using matched software, and displaying different genotypes based on the difference of Tm values displayed by hybridization peaks formed by different channels. The peak at rs1799853 locus appeared at 63 ℃ and was CC genotype, and at 57 ℃ it was TT genotype, and at both positions, the peak was CT genotype (FIG. 2). The peak at rs1057910 locus appeared at 54 ℃ as AA genotype, 60 ℃ as CC genotype, and both positions had peaks as AC genotype (FIG. 3). The peak at rs9923231 locus appeared at 68 ℃ and was the GG genotype, the AA genotype at 63 ℃, and the peak at both positions was the AG genotype (FIG. 4). As shown in fig. 5, the melting curve of the multi-sample is shown, and the success rate of typing reaches 100%.

4. The genomic DNA of 50 oral epithelial cells was subjected to sanger sequencing at the same time, and the detection results completely coincided with those of the present invention, thereby demonstrating that the method of the present invention has high accuracy in the results of polymorphisms of CYP2C9 x 2, CYP2C9 x 3 and VKORC1 genes.

Sequence listing

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Claims (7)

1. A probe for detecting polymorphism of CYP2C9 gene and VKORC1 gene, wherein the sequence of the probe is shown as SEQ ID No. 1, SEQ ID No. 4 or SEQ ID No. 7; or the two ends of the sequences of SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 7 are modified by groups.

2. The probe of claim 1, wherein the modifying group of SEQ ID No. 1 is: the modifying group of 5' is a fluorescent group HEX; the modifying group of the 3' is a quenching group BHQ 1; wherein the modifying group of SEQ ID No. 4 is: the modifying group of 5' is a fluorescent group FAM; the modifying group of the 3' is a quenching group BHQ 1; wherein the modifying group of SEQ ID No. 7 is: the modification group of 5' is a fluorescent group CY 5; the modifying group at the 3' is a quenching group BHQ 2.

3. The probe of claim 1, wherein the probe comprises a loop sequence and a stem sequence, wherein the loop sequence of SEQ ID No. 1 is 6-26 bp; wherein the loop sequence of SEQ ID NO. 4 is 6-18 bp; wherein the loop sequence of SEQ ID NO. 7 is 6-18 bp.

4. The primer for detecting the polymorphism of the CYP2C9 gene and the VKORC1 gene is characterized in that the sequence of the primer is shown as SEQ ID NO. 2 and SEQ ID NO. 3; 5 and 6 in SEQ ID No.: 5; SEQ ID No. 8 and SEQ ID No. 9.

5. Kit for detecting polymorphisms in the CYP2C9 gene and the VKORC1 gene, characterized in that the kit comprises the probe according to any one of claims 1 to 3 and/or the primer according to claim 4.

6. The kit of claim 5, wherein the kit further comprises Taq enzyme, dNTP, and/or Mg²⁺And instructions for use.

7. A method for detecting polymorphisms in the CYP2C9 gene and the VKORC1 gene, the method comprising the steps of:

(1) collecting a sample and extracting DNA;

(2) performing a fluorescent quantitative PCR reaction using the probe of any one of claims 1 to 3 and the primer of claim 4;

(3) and (3) analyzing the result by using a PCR instrument matched software, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks.

Images