Primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing

A pyrosequencing method, VKORC1C1173T technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of low sensitivity of VKORC1 genotyping, long detection cycle, cumbersome operation, etc., and achieve high-throughput sample operation The effect of detection, short reaction time, and simple sample handling

Inactive Publication Date: 2015-12-16
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the technical problems of low sensitivity, long detection period, cumbersome operation and high cost in the detection of VKORC1 genotyping by the above method, the present invention provides a

Method used

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  • Primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing
  • Primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing
  • Primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: the preparation of kit

[0068] 1. Design and synthesis of primers and probes

[0069] For the polymorphic sites VKORC1G1639A and VKORC1C1173T alleles of the human VKORC1 gene, specific mutation sites were selected, and primers were designed using PyroMarkAssayDesign2.0 software; the amplification primers and sequencing primers were first purified by PAGE, and then purified by HPLC. Wherein the 5' ends of SEQ ID NO.1 and SEQ ID NO.5 are biotinylated.

[0070] Table 1. Mutation site and type:

[0071] Mutation

[0072] The amplified sequence is shown in Table 2:

[0073] Table 2. Specific amplification primers and primer sequences

[0074]

[0075] 2. Selection of reference substance

[0076] A synthetic oligonucleotide chain TAYGGTTTGCAcontrololigo was used as the quality control substance; DNase / RNase-free water was used as the blank control substance.

[0077] 3. Composition of PCR reaction solution

[0078] Table 3. Composition of PCR...

Embodiment 2

[0083] Embodiment 2: the use of kit

[0084] 1. Sample testing

[0085] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, uracil DNA glycosylase, and TaqDNA polymerase, aliquot the system, add sample DNA, blank control substance or positive control substance as templates, and form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0086] The main components of VKORC1G1639A and VKORC1C1173T systems are as follows:

[0087] Table 5. Main components of VKORC1G1639A system

[0088]

[0089]

[0090] Table 6. Main components of VKORC1C1173T system

[0091]

[0092] The system reaction procedure is as follows:

[0093] Table 7. PCR reaction program

[0094]

[0095] After the amplification is completed, check the PCR results on agarose gel to proceed to the n...

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Abstract

The invention relates to a primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing, belonging to the technical field of in vitro nucleic acid detection. The primer pair comprises VKORC1 G1639A and VKORC1 C1173T forward amplification primers, VKORC1 G1639A and VKORC1 C1173T reverse amplification primers and VKORC1 G1639A and VKORC1 C1173T sequencing primers, wherein 5' terminals of the VKORC1 G1639A forward amplification primer and the VKORC1 C1173T reverse amplification primer are respectively subjected to biotin labelling. The kit comprises the amplification primers, a PCR (polymerase chain reaction) liquid 1, a PCR liquid 2, the sequencing primers, uracil DNA (deoxyribonucleic acid)glycosylase and Taq polymerase. The kit provided by the invention has the advantages of accurate detection results, high specificity, short detection period, simplicity in operation, capability of effectively meeting the requirements of clinical examination, capability of monitoring the reaction process in real time, short reaction time, sequencing of PCR products on a pyrosequencing instrument after the PCR products are simply treated, high throughput sample detection and higher sensitivity than gold standard methods, namely capillary electrophoresis sequencing methods.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a pair of primers and a kit for detecting VKORC1 genotyping by pyrosequencing. Background technique [0002] Vitamin K epoxide reductase complex subunit 1 (Vitamin Kepoxide reductase complex subunit 1, VKORC1) is a key enzyme in the metabolic cycle of vitamin K. Warfarin blocks vitamin K from participating in the catalytic reaction of carboxylase in the form of a cofactor due to the inhibition of this enzyme, inhibiting It inhibits the functional activity of coagulation factors II, VII, IX, and X, thereby producing an anticoagulant effect. A large number of studies at home and abroad have found that VKORC1 gene mutations will increase the sensitivity of the enzyme to warfarin, thereby enhancing the anticoagulant effect. The more common mutations include the G1639A mutation in the promoter region and the C1173T mutation in intron 1. In addition, studies ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869
Inventor 滕祥云朱江
Owner CHANGSHA 3G BIOTECH
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