CN111172253A - Kit and method for detecting CYP2C9 and VKORC1 gene polymorphism in one tube manner - Google Patents
Kit and method for detecting CYP2C9 and VKORC1 gene polymorphism in one tube manner Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting CYP2C9 and VKORC1 gene polymorphism in a tubular mode, wherein the kit comprises CYP2C91075A > C and VKORC11639G > A combined detection reaction liquid. Experiments prove that the primer and the probe in the combined detection reaction liquid have strong specificity, can realize direct saliva/blood sample PCR amplification analysis, can realize typing detection of two sites in one reaction tube, not only obviously improves the detection efficiency, but also obviously shortens the detection period, and has the advantages of good sensitivity, high specificity, accuracy, reliability, convenient and quick operation and the like.
Description
Technical Field
The invention relates to a kit and a method for detecting polymorphism of CYP2C9 and VKORC1 genes in a tubular mode, and belongs to the technical field of in vitro gene detection.
Background
Warfarin is a dicoumarol derivative oral anticoagulant widely used clinically, and the effectiveness and safety of anticoagulant treatment become important factors influencing the success of treatment of patients. The effective treatment range of warfarin is narrow, the influence factors of the curative effect are complex, and obvious individual difference and ethnic difference exist between dosage and effect. The average dose of the Huanggeng warfarin is about 3.0mg d-1About 4.5 mg. d for caucasian-1About 5.7 mg. d for melanoderm-1. Insufficient warfarin dosage is easy to form thrombus, and excessive warfarin dosage can increase bleeding risk. It is estimated that 15.2% of patients taking warfarin develop a blood side effect each year, with fatal major bleedingAccounting for 3.5%, the difference of the warfarin stable dose among different individuals can reach more than 20 times, which makes the warfarin dose not easy to be mastered clinically.
Warfarin is racemic isomer composed of S-warfarin and R-warfarin, wherein the anticoagulation activity of S-warfarin is about 5 times of that of R-warfarin, and 60% -70% of the anticoagulation activity of warfarin is provided. Warfarin is ineffective in anticoagulation in vitro, after oral absorption, inactive oxidized vitamin K cannot be converted into active reduced vitamin K by inhibiting oxidoreductase of liver vitamin K, the cyclic utilization of the vitamin K is blocked, the synthesis of vitamin K-dependent blood coagulation factors II, VII, IX and X is interfered, the gamma carboxylation of glutamic acid residue at the amino terminal of the blood coagulation factors is blocked, and the blood coagulation factors stay at inactive precursor stage to achieve the anticoagulation purpose.
Pharmacogenomics studies show that: the CYP2C9, VKORC1 and CYP4F2 gene polymorphisms are three major contributors to individual dose variation in warfarin, accounting for approximately 12%, 27% and 1-10% dose variation, respectively. The CYP2C9 gene has high genetic polymorphism, and wild-type CYP2C9 x 1 and mutant CYP2C9 x 2 to 13 exist, wherein the genes which are most closely metabolized by warfarin are mutant CYP2C9 x 2 and CYP2C9 x 3. CYP2C9 x 2 and CYP2C9 x 3 mutations occur less frequently in asians within the chinese han population, predominantly CYP2C9 x 3 mutants, ranging from 2% to 4%, whereas CYP2C9 x 2 mutants are rarely found. When CYP2C9 gene has CYP2C9 x 3 subtype, the enzyme activity is reduced by 80% compared with wild type. Research also shows that S-warfarin is metabolized by CYP2C9, and the mutant thereof greatly reduces the rate of warfarin metabolism, so that the drug-administered individual has higher bleeding risk in the early stage of use.
In addition, warfarin is a specific inhibitor of vitamin K epoxide reductase VKORC 1. A great deal of research shows that the gene polymorphism (-1639G/A) of the VKORC1 promoter is the most main factor influencing the ethnic difference and the individual difference in the warfarin demand dose. Compared with the patients carrying the-1639 AA allele, the average daily dose of warfarin of the patients carrying the-1639 GA allele is increased by 61 percent, and the explanation strength of the warfarin dose is obviously higher than that of CYP2C 9.
Based on this, the American FDA revised the warfarin pharmacy Specification at 8 months of 2007 (Coumadin, product of Bai Shi Guibao Co., Ltd.), suggesting that the CYP2C9 and VKORC1 gene polymorphisms have an influence on the warfarin therapeutic effect, and it is recommended to detect CYP2C9 and VKORC1 genotypes before administration. In 2009 the FDA again modified the warfarin dosing label, suggesting that the initial dose was determined based on the patient VKORC1 and CYP2C9 genotypes, and provided the following dose recommendations:
practice proves that warfarin individual administration is carried out according to the genotype of a patient and by combining clinical information of the patient, the anticoagulation standard-reaching rate of warfarin can be obviously improved, anticoagulation complications are reduced, and the hospitalization rate of the patient is reduced. Therefore, it is recommended to test CYP2C9 x 3 and VKORC1 genes prior to warfarin administration, and to direct warfarin dosing according to patient genotype.
The current detection methods for gene polymorphism mainly comprise: PCR-RFLP method, PCR-chip hybridization method, PCR-sequencing method and Taqman-qPCR method, wherein: the PCR-RFLP method mainly depends on restriction enzyme cutting sites of a target, the experiment difficulty is increased, and meanwhile, the subsequent electrophoretic analysis is easy to cause laboratory pollution and cannot be applied clinically in a large scale; the PCR-chip hybridization method combines the PCR technology and the gene chip technology, has the same risks of complicated steps and cross contamination in laboratories, has low detection sensitivity and poor specificity, and is easy to generate false positive results; the PCR-sequencing method is a gold standard for gene detection, but the operation process is too long, the cost is too high, and meanwhile, the instrument is expensive and is difficult to be applied in a hospital on a large scale; the Taqman-qPCR method has high detection sensitivity, but the detection specificity is not high due to site sequence, and the Taqman-qPCR method is used for typing through a Genotyping method, has certain requirements on the number of samples and the polymorphism distribution, and is not suitable for detecting sites with too low mutation frequency and a small amount of samples. Therefore, there is a need in the art for a technique for detecting polymorphisms of CYP2C9 and VKORC1 gene which is rapid, accurate, sensitive, highly specific, and easy to operate.
Disclosure of Invention
In view of the above problems and needs in the prior art, the present invention provides a kit and a method for one-tube detection of polymorphisms of CYP2C9 and VKORC1 genes, which are rapid, accurate, sensitive, specific and easy to operate.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a kit for detecting CYP2C9 and VKORC1 gene polymorphism in a single tube mode comprises CYP2C91075A > C and VKORC11639G > A combined detection reaction liquid; the CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction liquid comprises a primer group aiming at a sequence shown by SEQ ID NO.1 and SEQ ID NO.2 of a CYP2C91075A & gtC site, a probe group aiming at a sequence shown by SEQ ID NO.3 and SEQ ID NO.4, a primer group aiming at a sequence shown by SEQ ID NO.5 and SEQ ID NO.6 of a VKORC11639G & gtA site and a probe group aiming at a sequence shown by SEQ ID NO.7 and SEQ ID NO. 8.
Further, all probes are labeled with a reporter fluorescent dye at the 5 'end and a quencher fluorescent dye at the 3' end.
Further, 5 'ends of all the probes are labeled with any one of the reporter fluorescent dyes FAM, VIC, ROX and CY5, and 3' ends of all the probes are labeled with the quenching fluorescent dye BHQ.
Furthermore, the 5 'end of the wild-type probe aiming at the CYP2C91075A & gtC site is marked with a reporter fluorescent dye FAM, and the 5' end of the mutant-type probe aiming at the CYP2C91075A & gtC site is marked with a reporter fluorescent dye VIC.
Further, the 5 'end of the wild-type probe for VKORC11639G > A site is labeled with a reporter fluorescent dye ROX, and the 5' end of the mutant probe for VKORC11639G > A site is labeled with a reporter fluorescent dye CY 5.
Furthermore, the CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction solution also comprises a modified HotStar Taq enzyme, a direct amplification PCR amplification buffer solution, a direct amplification reagent and a stabilizing agent.
Further, the composition of the direct amplification PCR amplification buffer solution is as follows: Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride) buffer solution containing 200mmol/L to 400mmol/L KCl (potassium chloride).
Further, the composition of the direct amplification reagent is as follows: 25 mmol/L-50 mmol/L Tris-HCl (Tris hydroxymethyl aminomethane hydrochloride), 1 mmol/L-5 mmol/L EDTA (ethylene diamine tetraacetic acid), 200 mmol/L-400 mmol/L NaCl (sodium chloride), 0.1 wt% -0.6 wt% SDS (sodium dodecyl sulfate), 0.5 mol/L-2.0 mol/L sorbitol, 0.5 mmol/L-0.8 mmol/L DTT (dithiothreitol).
Further, the stabilizer is composed of: 0.5-1.0 wt% of glycerol, 0.2-0.6 wt% of NP40 (ethyl phenyl polyethylene glycol), 0.2-0.6 wt% of tween20 (Tween 20), and 0.5-2 mg/mL of BSA (bovine serum albumin).
Further, the kit also comprises a positive control substance and a negative control substance, wherein the positive control substance adopts human genome, and the negative control substance adopts DEPC H2O。
A method for detecting CYP2C9 and VKORC1 gene polymorphism in a tubular mode comprises the kit disclosed by the invention, and the method comprises the following steps:
a) directly adding a sample to be detected into a PCR reaction tube containing CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction liquid, uniformly mixing and centrifuging, and then putting into a fluorescent quantitative PCR instrument for PCR amplification reaction;
b) and carrying out fluorescence detection on the amplification product, and directly judging the polymorphism of the test sample according to the fluorescence curve and the Ct value.
Further, the sample to be detected is saliva or a blood sample.
Further, the sample to be detected in the PCR reaction tube is 1 muL, the combined detection reaction solution is 20 muL, and the concentration of the primer and the concentration of the probe in the combined detection reaction solution are both 200 nmol/L.
Further, the conditions for performing PCR amplification reaction in step a) are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10 seconds; annealing at 60 ℃ for 30 seconds; 40 cycles.
Compared with the prior art, the invention has the following beneficial technical effects:
1. experiments prove that the primers and the probes designed by the invention have strong specificity, can realize direct adoption of saliva/blood samples for PCR amplification analysis, omit complicated nucleic acid extraction and purification steps, and obviously save experiment consumables and labor cost;
2. the invention can realize the typing detection of two sites in one reaction tube, not only obviously improves the detection efficiency, but also obviously shortens the detection period, and can directly carry out result interpretation according to the fluorescence curve, and the interpretation result is simple, convenient and objective;
3. the CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction solution can ensure the accuracy and sensitivity of the experiment, and the consistency of the detection result is 100 percent compared with that of a gold standard first-generation sequencing method.
Drawings
FIG. 1 is a fluorescent PCR profile of a CYP2C91075A > C wild-type sample;
FIG. 2 is a fluorescent PCR profile of a CYP2C91075A > C mutant sample;
FIG. 3 is a fluorescent PCR plot of a CYP2C91075A > C heterozygous sample;
FIG. 4 is a graph of fluorescence PCR profiles of VKORC11639G > A wild type samples;
FIG. 5 is a fluorescent PCR plot of VKORC11639G > A mutant samples;
FIG. 6 is a fluorescent PCR plot of VKORC11639G > A heterozygous samples.
Detailed Description
The technical solutions and effects of the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. The reagents and biomaterials used below were all commercial products unless otherwise specified.
Example 1: the kit for detecting CYP2C9 and VKORC1 gene polymorphism in one tube type is prepared
Firstly, primers and probes aiming at CYP2C91075A & gtC and VKORC11639G & gtA are synthesized respectively according to the following table:
secondly, preparing a CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction solution according to the following table, wherein the reaction solution comprises:
| components | Proportioning |
| Upstream primer of sequence shown in SEQ ID NO.1 | 200nmol/L |
| Downstream primer of sequence shown in SEQ ID NO.2 | 200nmol/L |
| Wild type probe of sequence shown in SEQ ID NO.3 | 200nmol/L |
| Mutant probe of sequence shown in SEQ ID NO.4 | 200nmol/L |
| Upstream primer of sequence shown in SEQ ID NO.5 | 200nmol/L |
| Downstream primer of sequence shown in SEQ ID NO.6 | 200nmol/L |
| Wild type probe of sequence shown in SEQ ID NO.7 | 200nmol/L |
| Mutant probe of sequence shown in SEQ ID NO.8 | 200nmol/L |
In addition, the kit also comprises an improved HotStar Taq enzyme, a direct amplification PCR amplification buffer solution, a direct amplification reagent and a stabilizer, wherein the direct amplification PCR amplification buffer solution comprises the following components: Tris-HCl buffer solution, which contains 200 mmol/L-400 mmol/LKCl; the direct amplification reagent comprises the following components: 25 mmol/L-50 mmol/L Tris-HCl (Tris hydroxymethyl aminomethane hydrochloride), 1 mmol/L-5 mmol/L EDTA (ethylene diamine tetraacetic acid), 200 mmol/L-400 mmol/L NaCl (sodium chloride), 0.1 wt% -0.6 wt% SDS (sodium dodecyl sulfate), 0.5 mol/L-2.0 mol/L sorbitol, 0.5 mmol/L-0.8 mmol/L LDTT (dithiothreitol); the stabilizer comprises the following components: 0.5-1.0 wt% of glycerol, 0.2-0.6 wt% of NP40 (ethyl phenyl polyethylene glycol), 0.2-0.6 wt% of tween20 (Tween 20), and 0.5-2 mg/mL of BSA (bovine serum albumin).
Three, assemble kit
The kit comprises 1 PCR reaction tube, wherein CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction liquid is filled in the PCR reaction tube;
in addition, the kit also comprises 1 tube of positive control substance and 1 tube of negative control substance, wherein the positive control substance adopts human genome, and the negative control substance adopts DEPC H2O。
Example 2
The method for detecting the polymorphism of CYP2C9 and VKORC1 genes by using the kit comprises the following steps:
a) taking 1 μ L of saliva (or blood sample) sample to be detected, directly adding the saliva sample into a PCR reaction tube containing 20 μ L of CYP2C91075A > C and VKORC11639G > A combined detection reaction solution, mixing uniformly, centrifuging, and then putting the saliva sample into a fluorescent quantitative PCR instrument for PCR amplification reaction: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10 seconds; annealing at 60 ℃ for 30 seconds; 40 cycles;
b) and (3) carrying out fluorescence detection on the amplification product, and directly interpreting the polymorphism of the test sample according to fluorescence curves and Ct values of four channels of FAM, VIC, ROX and CY5, namely:
if FAM fluorescence signal is on line but VIC fluorescence signal is not on line (as shown in FIG. 1), the site CYP2C91075A > C is judged to be wild type; if the FAM fluorescence signal is not on line but the VIC fluorescence signal is on line (as shown in FIG. 2), the CYP2C91075A & gtC site is judged to be the mutant type; if the FAM fluorescence signal and the VIC fluorescence signal both start lines (as shown in FIG. 3), the site CYP2C91075A & gtC is judged to be a heterozygote; if the ROX fluorescence signal is on line but the CY5 fluorescence signal is not on line (as shown in FIG. 4), the site VKORC11639G > A is judged to be wild type; if the ROX fluorescence signal is not on line but the CY5 fluorescence signal is on line (as shown in FIG. 5), the site VKORC11639G > A is judged to be mutant; when both the ROX fluorescence signal and the CY5 fluorescence signal appear on line (as shown in FIG. 6), it is judged that the VKORC11639G > A site is heterozygous.
Also, as can be seen in connection with fig. 1 to 6: by adopting the CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction solution, the genotype with good amplification can be obtained, and non-specific amplification is avoided.
In addition, the inventor also proves that:
according to the kit and the method, positive plasmids synthesized by gene sequences of other sections except CYP2C91075A & gtC and VKORC11639G & gtA gene detection sites and genomic DNA of staphylococcus aureus and escherichia coli are not crossed, and the designed primers and probes have strong specificity;
the minimum detection limit of each type of plasmid sample by the kit and the method is 1 x 102The copies/mu L is 0.1 ng/mu L of the lowest detection of each type of human genome DNA samples, and the sensitivity is high;
the detection results of the adopted positive reference substances are positive, the positive coincidence rate is 100%, and the accuracy is good;
using precision reference (1X 10)5copies/. mu.L) (n is 10), the coefficient of variation (CV,%) of Ct value is less than 5.0%, and the precision is very high.
In conclusion, the kit and the method have the remarkable progresses of good sensitivity, high specificity, accuracy, reliability, convenience and quickness in operation and the like.
It is finally necessary to point out here: the above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (10)
1. A kit for detecting CYP2C9 and VKORC1 gene polymorphism in a tubular mode is characterized in that: comprises CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction liquid; the CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction solution comprises a primer group aiming at a sequence shown by SEQ ID NO.1 and SEQ ID NO.2 of a CYP2C91075A & gtC site, a probe group aiming at a sequence shown by SEQ ID NO.3 and SEQ ID NO.4, a primer group aiming at a sequence shown by SEQ ID NO.5 and SEQ ID NO.6 of a VKORC11639G & gtA site and a probe group aiming at a sequence shown by SEQ ID NO.7 and SEQ ID NO. 8.
2. The kit of claim 1, wherein: the 5 'ends of all the probes are marked with any one of reporting fluorescent dyes FAM, VIC, ROX and CY5, and the 3' ends of all the probes are marked with quenching fluorescent dyes BHQ.
3. The kit of claim 2, wherein: the 5 'end of a wild-type probe aiming at CYP2C91075A & gtC site is marked with a reporter fluorescent dye FAM, the 5' end of a mutant-type probe aiming at CYP2C91075A & gtC site is marked with a reporter fluorescent dye VIC, the 5 'end of a wild-type probe aiming at VKORC11639G & gtA site is marked with a reporter fluorescent dye ROX, and the 5' end of a mutant-type probe aiming at VKORC11639G & gtA site is marked with a reporter fluorescent dye CY 5.
4. The kit of claim 1, wherein: the CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction solution further comprises an improved HotStar Taq enzyme, a direct amplification PCR amplification buffer solution, a direct amplification reagent and a stabilizer.
5. The kit according to claim 4, wherein the composition of the direct amplification PCR amplification buffer is: Tris-HCl buffer solution, which contains 200 mmol/L-400 mmol/L KCl.
6. The kit according to claim 4, wherein the composition of the direct amplification reagent is as follows: 25 mmol/L-50 mmol/L Tris-HCl, 1 mmol/L-5 mmol/L EDTA, 200 mmol/L-400 mmol/L NaCl, 0.1 wt% -0.6 wt% SDS, 0.5 mol/L-2.0 mol/L sorbitol, 0.5 mmol/L-0.8 mmol/L DTT.
7. The kit according to claim 4, characterized in that the composition of the stabilizer is: 0.5-1.0 wt% of glycerol, 0.2-0.6 wt% of NP40, 0.2-0.6 wt% of tween20 and 0.5-2 mg/mL of mLBSA.
8. The kit of claim 1, wherein: the kit also comprises a positive control substance and a negative control substance, wherein the positive control substance adopts human genome, and the negative control substance adopts DEPC H2O。
9. A method for one-tube detection of polymorphisms of CYP2C9 and VKORC1 genes, comprising the kit of any one of claims 1 to 8, and the method comprising the steps of:
a) directly adding a sample to be detected into a PCR reaction tube containing CYP2C91075A & gtC and VKORC11639G & gtA combined detection reaction liquid, uniformly mixing and centrifuging, and then putting into a fluorescent quantitative PCR instrument for PCR amplification reaction: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10 seconds; annealing at 60 ℃ for 30 seconds; 40 cycles;
b) and carrying out fluorescence detection on the amplification product, and directly judging the polymorphism of the test sample according to the fluorescence curve and the Ct value.
10. The method of claim 9, wherein: the sample to be detected in the PCR reaction tube is 1 mu L, the combined detection reaction solution is 20 mu L, and the concentrations of the primer and the probe in the combined detection reaction solution are both 200 nmol/L.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293920A (en) * | 2014-09-17 | 2015-01-21 | 武汉康录生物技术有限公司 | Kit for quickly detecting polymorphism of human VKORC1 and CYP2CP genes and using method of kit |
| CN106702005A (en) * | 2017-03-01 | 2017-05-24 | 深圳荻硕贝肯精准医学有限公司 | Primers, probes, kit and method for testing human VKORC1 and CYP2C9 gene polymorphisms |
| CN107841540A (en) * | 2017-12-13 | 2018-03-27 | 益善生物技术股份有限公司 | A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit |
| CN108251510A (en) * | 2018-03-13 | 2018-07-06 | 普迈德（北京）科技有限公司 | A kind of kit, detection method and its application of folic acid metabolism ability Genotyping |
| CN109609627A (en) * | 2019-01-30 | 2019-04-12 | 上海酷乐生物科技有限公司 | A kind of detection kit and detection method of direct expansion type MTHFR and/or MTRR and MTR gene pleiomorphism |
-
2020
- 2020-02-25 CN CN202010116817.0A patent/CN111172253A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293920A (en) * | 2014-09-17 | 2015-01-21 | 武汉康录生物技术有限公司 | Kit for quickly detecting polymorphism of human VKORC1 and CYP2CP genes and using method of kit |
| CN106702005A (en) * | 2017-03-01 | 2017-05-24 | 深圳荻硕贝肯精准医学有限公司 | Primers, probes, kit and method for testing human VKORC1 and CYP2C9 gene polymorphisms |
| CN107841540A (en) * | 2017-12-13 | 2018-03-27 | 益善生物技术股份有限公司 | A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit |
| CN108251510A (en) * | 2018-03-13 | 2018-07-06 | 普迈德（北京）科技有限公司 | A kind of kit, detection method and its application of folic acid metabolism ability Genotyping |
| CN109609627A (en) * | 2019-01-30 | 2019-04-12 | 上海酷乐生物科技有限公司 | A kind of detection kit and detection method of direct expansion type MTHFR and/or MTRR and MTR gene pleiomorphism |
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