Pyrosequencing primer pair and kit for qualitatively detecting CYP2D6 genotyping

A technology of CYP2D6C188T and pyrosequencing, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of low sensitivity of CYP2D6 genotyping, long detection cycle, cumbersome operation, etc. Achieve high-throughput sample detection, short reaction time, and simple sample processing

Inactive Publication Date: 2016-12-21
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to solve the technical problems of low sensitivity, long detection cycle, cumbersome operation and high cost in the detection of CYP2D6 genotyping by the above method, the present invention provides a method with high sensitivity, strong specificity, short detection cycle, simple operation and Pyrosequencing primer pair and kit for qualitative detection of CYP2D6 genotyping that effectively meet clinical testing requirements

Method used

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  • Pyrosequencing primer pair and kit for qualitatively detecting CYP2D6 genotyping
  • Pyrosequencing primer pair and kit for qualitatively detecting CYP2D6 genotyping
  • Pyrosequencing primer pair and kit for qualitatively detecting CYP2D6 genotyping

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1: the preparation of kit

[0078] 1. Design and synthesis of primers and probes

[0079] For the polymorphic sites CYP2D6C188T and CYP2D6G4268C alleles of the human CYP2D6 gene, select specific mutation sites, and use PyroMark Assay Design2.0 software to design primers; the amplification primers and sequencing primers are first purified by PAGE, and then HPLC Purified, wherein the 5' ends of SEQ ID NO.1 and SEQ ID NO.4 were biotin-labeled.

[0080] Table 1. Mutation site and type

[0081] Mutation

[0082] The amplified sequence is shown in Table 2:

[0083] Table 2. Specific amplification primers and primer sequences

[0084]

[0085]

[0086] 2. Selection of reference substance

[0087] A synthetic oligonucleotide chain TAYGGTTTGCA control oligo was used as the quality control product; DNase / RNase-Free water was used as the blank control product.

[0088] 3. Composition of PCR reaction solution

[0089] Table 3. Composition of PCR rea...

Embodiment 2

[0094] Embodiment 2: the use of kit

[0095] 1. Sample testing

[0096] 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.

[0097] The main components of CYP2D6C188T and CYP2D6G4268C systems are as follows:

[0098] Table 5. Main components of CYP2D6C188T system

[0099]

[0100] Table 6. Main components of CYP2D6G4268C system

[0101]

[0102] The system reaction procedure is as follows:

[0103] Table 7. PCR reaction program

[0104]

[0105]

[0106] After the amplification is completed, check the PCR results on agarose gel to proceed to the nex...

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Abstract

The invention relates to a pyrosequencing primer pair for qualitatively detecting CYP2D6 genotyping; the primer pair includes a forward amplification primer and a reverse amplification primer, and a sequencing primer, and 5' ends of the forward amplification primer and reverse amplification primer are subjected to biotin labeling respectively; the invention also relates to a pyrosequencing kit for qualitatively detecting CYP2D6 genotyping; the kit includes amplification primers, PCR (polymerase chain reaction) liquid 1, PCR liquid 2, sequencing primers, uracil DNA glycosylase and Taq polymerase. The pyrosequencing primer pair and kit have the advantages that detection results are accurate, the specificity is high, detection period is short, operation is simple and clinical examination requirements can be effectively met and additionally have the advantages that reaction process can be monitored in real time, reaction time is short, PCR products can be fed to a pyrosequencing instrument for sequencing and high-throughput sample detection just through simple treatment, and the sensitivity is higher than that of a golden standard method, namely capillary electrophoresis sequencing method.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a pair of pyrosequencing primers and a kit for qualitative detection of CYP2D6 genotyping. Background technique [0002] Cytochrome P450 2D6 (Cytochrome P450 2D6, CYP2D6) is a very important drug metabolizing enzyme in the CYP enzyme system. In the CYP450 superfamily, CYP2 is the largest family, which can be divided into five subfamilies in humans, numbered A~E. CYP2D is located on chromosome 22 and consists of CYP2D6, CYP2D7P and CYP2D8P, among which CYP2D7P and CYP2D8P are pseudogenes and are not expressed. As a complete functional gene, CYP2D6 contains 9 exons and 8 introns, with a total length of about 7kb. CYP2D6 is the earliest discovered P450 enzyme with gene polymorphism. At present, more than 70 mutant genes of CYP2D6 have been found, of which more than 15 are translated into inactive or non-enzymatic codes, and others are translated into reduce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2565/301C12Q2535/122
Inventor 滕祥云辛盛
Owner CHANGSHA 3G BIOTECH
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