Multiplex PCR combined pyrosequencing kit used for guiding individualized medication of warfarin and detection method thereof

Abstract

The invention provides a multiplex PCR combined pyrosequencing kit used for guiding individualized medication of warfarin and a detection method thereof, relating to warfarin. The kit comprises a whole blood genome DNA extraction reagent, multiplex PCR amplification primers, a multiplex PCR amplification reagent, a single-stranded DNA separating and purifying reagent, pyrosequencing primers, a pyrosequencing reagent and a kit body. The detection method comprises the following steps: extraction of human whole blood genome DNA; multiplex PCR amplification; separation and purification of a single-stranded DNA sample; and pyrosequencing and result analysis. According to the invention, main factors influencing difference of individual dosage of warfarin, i.e., important gene polymorphic sites of VKORC1 and CYP2C9, specifically being VKORC1-1639G>A, CYP2C9*2 and CYP2C9*3, are detected so as to guide individualized dosage of warfarin in treatment of patients needing to take warfarin, thereby achieving the purpose of reduction in side effects.

Description

technical field

[0001] The invention belongs to the field of molecular biology testing and relates to warfarin, in particular to a multiplex PCR combined pyrosequencing kit and a detection method for guiding warfarin individualized medication. Background technique

[0002] Warfarin is a dicoumarin derivative that exerts its anticoagulant effect by inhibiting the interconversion of vitamin K and its 2,3-epoxide (vitamin K epoxide). Although the oldest oral anticoagulant, warfarin is still the most commonly prescribed drug for patients requiring long-term anticoagulant therapy [1] . Clinical applications of warfarin include primary and secondary prevention of venous thromboembolic disease (VTE), prevention of thromboembolism in atrial fibrillation (AF), valvular disease, prosthetic valve replacement and prevention of intracardiac thrombosis, etc. [2,3] . Meta-analysis of nonvalvular atrial fibrillation studies shows that warfarin can reduce the relative risk of stroke by 64...

Examples

Embodiment 1

[0068] The key primer components and sequences of the kit are as follows:

[0069] PCR Primer 1 (Seq NO1):

[0070] VKORC1-1639G>A (rs9923231) upstream primer 5'-GGGAAGTCAAGCAAGAGAAGACCT-3';

[0071] PCR primer 2 (Seq NO2):

[0072] VKORC1-1639G>A (rs9923231) downstream primer 5'-GCCTCCAGGGTTCAAGTGG-3';

[0073] PCR primer 3 (Seq NO3):

[0074] CYP2C9*2 (rs1799853) upstream primer 5'-TACAGAGCTCCTCGGGCAGA-3';

[0075] PCR primer 4 (Seq NO4):

[0076] CYP2C9*2 (rs1799853) downstream primer 5'-CAGCGGGCTTCCTCTTGA-3';

[0077] PCR primer 5 (Seq NO5):

[0078] CYP2C9*3 (rs1057910) upstream primer 5'-TAAAGTCCAGGAAGAGATTGAACG-3';

[0079] PCR primer 6 (Seq NO6):

[0080] CYP2C9*3 (rs1057910) downstream primer 5'-GGGGACTTCGAAAACATGG-3';

[0081] Sequencing primer 1 (Seq NO7):

[0082] VKORC1-1639G>A (rs9923231) sequencing primer 5'-CCTGAAAAACAACCATTG-3';

[0083] Sequencing primer 2 (Seq NO8):

[0084] CYP2C9*2 (rs1799853) sequencing primer 5'-GAGGAGCATTGAGGAC-3';

[0085] S...

Embodiment 2

[0207] Take 2 mL of peripheral venous blood from patient B who is planning to choose warfarin as long-term primary anticoagulant prophylaxis for atrial fibrillation, and use the kit and method of the present invention to detect warfarin individualized drug genes. All operations are the same as in Example 1, and the test result of this patient is VKORC1-1639G>A. Figure 6 For G / A genotype, CYP2C9*2 as Figure 8 is C / C genotype,

[0208] CYP2C9*3 as Figure 9It is A / A genotype, combined with the patient's age of 45 years, height of 175cm, weight of 75kg and the situation of combined use of amiodarone, the recommended warfarin for this patient can be obtained according to the prediction formula of IWPC combined with International Warfarin Pharmacogenomics The dosage is 30mg / week.

Embodiment 3

[0210] Take 2 mL of peripheral venous blood from patient C who intends to choose warfarin anticoagulant therapy for deep vein thrombosis, and use the kit and method of the present invention to detect the gene of warfarin individualized drug use.

[0211] All operations are the same as in Example 1, and the test result of this patient is VKORC1-1639G>A. Figure 7 For A / A genotype, CYP2C9*2 as Figure 8 For C / C genotype, CYP2C9*3 as Figure 10 It is A / C genotype, combined with the patient's age of 70 years, height of 158cm, weight of 70kg and the situation of not using amiodarone and other drugs in combination, according to the prediction formula of IWPC combined with international warfarin pharmacogenomics, the recommendation of this patient can be obtained The dose of warfarin was 11mg / week.

technical field

[0001] The invention belongs to the field of molecular biology testing and relates to warfarin, in particular to a multiplex PCR combined pyrosequencing kit and a detection method for guiding warfarin individualized medication. Background technique

[0002] Warfarin is a dicoumarin derivative that exerts its anticoagulant effect by inhibiting the interconversion of vitamin K and its 2,3-epoxide (vitamin K epoxide). Although the oldest oral anticoagulant, warfarin is still the most commonly prescribed drug for patients requiring long-term anticoagulant therapy [1] . Clinical applications of warfarin include primary and secondary prevention of venous thromboembolic disease (VTE), prevention of thromboembolism in atrial fibrillation (AF), valvular disease, prosthetic valve replacement and prevention of intracardiac thrombosis, etc. [2,3] . Meta-analysis of nonvalvular atrial fibrillation studies shows that warfarin can reduce the relative risk of stroke by 64...

Examples

Embodiment 1

[0068] The key primer components and sequences of the kit are as follows:

[0069] PCR Primer 1 (Seq NO1):

[0070] VKORC1-1639G>A (rs9923231) upstream primer 5'-GGGAAGTCAAGCAAGAGAAGACCT-3';

[0071] PCR primer 2 (Seq NO2):

[0072] VKORC1-1639G>A (rs9923231) downstream primer 5'-GCCTCCAGGGTTCAAGTGG-3';

[0073] PCR primer 3 (Seq NO3):

[0074] CYP2C9*2 (rs1799853) upstream primer 5'-TACAGAGCTCCTCGGGCAGA-3';

[0075] PCR primer 4 (Seq NO4):

[0076] CYP2C9*2 (rs1799853) downstream primer 5'-CAGCGGGCTTCCTCTTGA-3';

[0077] PCR primer 5 (Seq NO5):

[0078] CYP2C9*3 (rs1057910) upstream primer 5'-TAAAGTCCAGGAAGAGATTGAACG-3';

[0079] PCR primer 6 (Seq NO6):

[0080] CYP2C9*3 (rs1057910) downstream primer 5'-GGGGACTTCGAAAACATGG-3';

[0081] Sequencing primer 1 (Seq NO7):

[0082] VKORC1-1639G>A (rs9923231) sequencing primer 5'-CCTGAAAAACAACCATTG-3';

[0083] Sequencing primer 2 (Seq NO8):

[0084] CYP2C9*2 (rs1799853) sequencing primer 5'-GAGGAGCATTGAGGAC-3';

[0085] S...

Embodiment 2

[0207] Take 2 mL of peripheral venous blood from patient B who is planning to choose warfarin as long-term primary anticoagulant prophylaxis for atrial fibrillation, and use the kit and method of the present invention to detect warfarin individualized drug genes. All operations are the same as in Example 1, and the test result of this patient is VKORC1-1639G>A. Figure 6 For G / A genotype, CYP2C9*2 as Figure 8 is C / C genotype,

[0208] CYP2C9*3 as Figure 9It is A / A genotype, combined with the patient's age of 45 years, height of 175cm, weight of 75kg and the situation of combined use of amiodarone, the recommended warfarin for this patient can be obtained according to the prediction formula of IWPC combined with International Warfarin Pharmacogenomics The dosage is 30mg / week.

Embodiment 3

[0210] Take 2 mL of peripheral venous blood from patient C who intends to choose warfarin anticoagulant therapy for deep vein thrombosis, and use the kit and method of the present invention to detect the gene of warfarin individualized drug use.

[0211] All operations are the same as in Example 1, and the test result of this patient is VKORC1-1639G>A. Figure 7 For A / A genotype, CYP2C9*2 as Figure 8 For C / C genotype, CYP2C9*3 as Figure 10 It is A / C genotype, combined with the patient's age of 70 years, height of 158cm, weight of 70kg and the situation of not using amiodarone and other drugs in combination, according to the prediction formula of IWPC combined with international warfarin pharmacogenomics, the recommendation of this patient can be obtained The dose of warfarin was 11mg / week.

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