Kit, method and application for detecting gene polymorphism based on shared primer probe

A gene polymorphism and kit technology, applied in the field of PCR detection of gene polymorphism, can solve the problems of detection time up to 4 to 5 hours, cumbersome operation, special equipment, PCR product contamination, etc.

Active Publication Date: 2020-06-02
ANNGEEN BIOTECHNOLOGY CO LTD
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AI Technical Summary

Problems solved by technology

Sequencing typing is the gold standard for detection in this field, but it has not been widely promoted due to the cumbersome operation and special equipment; the rest of the methods are derived from PCR amplification technology and are relatively widely used
Among them, the detection process of sequencing typing method includes PCR amplification and sequencing, and the operation is cumbersome. The detection time is generally as long as 4 to 5 hours, and the PCR reaction tube needs to be opened during the operation process, which is easy to cause contamination of PCR products; high resolution The characteristic of the melting curve typing method is to use the difference in Tm value caused by the difference of the base of the polymorphic site, and detect by identifying different Tm values. 1°C, so equipment that can accurately identify the temperature is needed. Such equipment is relatively rare, and most of them are imported and expensive. The detection process of gene chip typing method includes PCR amplification and probe hybridization. The operation is cumbersome, and the detection time is generally as long as 4 hours. ~5 hours, and the PCR reaction tube needs to be opened during the operation, which is likely to cause contamination of PCR products; the TaqMan probe typing method uses ARMS primers to identify polymorphic sites, and this method detects a polymorphic site Two reaction wells are required, and the throughput is relatively low; MGB probe typing uses MGB-labeled probes to identify polymorphic sites, MGB probe design is difficult and expensive, and finding a suitable MGB probe requires screening A lot of lines, the cost is higher

Method used

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  • Kit, method and application for detecting gene polymorphism based on shared primer probe
  • Kit, method and application for detecting gene polymorphism based on shared primer probe
  • Kit, method and application for detecting gene polymorphism based on shared primer probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1 Design optimization of primers, optimization screening of markers

[0136] 1. The design and screening of wild-type and mutant-type probe ARMS primers, the specific steps are as follows:

[0137] 1) Preliminarily design a series of ARMS primers for CYP2C19*2, CYP2C19*3, CYP2C19*17 wild-type and mutant loci, with a Tm value of 58-62°C, and the sequences are shown in Table 1:

[0138] Table 1. Preliminary design of CYP2C19*2, CYP2C19*3, CYP2C19*17 wild-type and mutant ARMS primers

[0139]

[0140]

[0141]

[0142] 2) CYP2C19*2 wild-type ARMS primers, downstream primers, and SYBR-premix buffer to prepare PCR reaction solution, respectively detect 5000 copy / μl of CYP2C19*2 wild-type plasmid and mutant plasmid; CYP2C19*2 mutant ARMS primers, downstream primers , SYBR-premix buffer to prepare PCR reaction solution, respectively detect 5000 copy / μl of CYP2C19*2 wild-type plasmid and mutant plasmid; CYP2C19*3 wild-type ARMS primer, downstream primer, SYBR-...

Embodiment 2

[0180] Example 2 Primer preparation and kit assembly

[0181] 1. Synthesis and fluorescent labeling of probe-based ARMS primers, quenching probes, and downstream primers in this application

[0182] According to Example 1, optimize and screen the probe ARMS primers, quencher probes, downstream primers and markers, and perform primer synthesis and fluorescent labeling. The specific markers are CYP2C19*2 wild-type probe ARMS primers 5′-end labeled FAM, CYP2C19*2 mutant probe ARMS primer 5′-end labeled TET, CYP2C19*2 quencher probe 3′-end labeled BHQ2, CYP2C19*2 downstream primer; CYP2C19*3 wild-type probe ARMS primer 5′-end labeled ROX , CYP2C19*3 mutant probe ARMS primer 5′-end labeled CY5, CYP2C19*3 quencher probe 3′-end labeled BHQ2, CYP2C19*3 downstream primer; CYP2C19*17 wild-type probe ARMS primer 5′-end labeled FAM, CYP2C19*17 mutant type probe ARMS primer 5′ end labeled TET, CYP2C19*17 quenching probe 3′ end labeled BHQ2, CYP2C19*17 downstream primer.

[0183] The spec...

Embodiment 3

[0224] Embodiment 3 detection sensitivity

[0225] Use the kit a for detecting human CYP2C19 gene polymorphisms based on shared primer probes prepared in Example 2 and the kit for detecting CYP2C19 gene polymorphisms based on ARMS primers + TaqMan probes b, and the detection of CYP2C19 gene polymorphisms based on MGB probes morphological kit c comparative test, the specific comparative test steps are as follows:

[0226] Step 1. Prepare sensitivity templates, including CYP2C19*2 homozygous wild type 2ng / μl, 1ng / μl, 0.5ng / μl, CYP2C19*2 homozygous mutant 2ng / μl, 1ng / μl, 0.5ng / μl, CYP2C19* 2 heterozygous 2ng / μl, 1ng / μl, 0.5ng / μl; CYP2C19*3 homozygous wild type 2ng / μl, 1ng / μl, 0.5ng / μl, CYP2C19*3 homozygous mutant 2ng / μl, 1ng / μl μl, 0.5ng / μl, CYP2C19*3 heterozygous 2ng / μl, 1ng / μl, 0.5ng / μl; CYP2C19*17 homozygous wild type 2ng / μl, 1ng / μl, 0.5ng / μl, CYP2C19*17 pure Synzygous mutant 2ng / μl, 1ng / μl, 0.5ng / μl, CYP2C19*17 heterozygous 2ng / μl, 1ng / μl, 0.5ng / μl;

[0227] Step 2, prepar...

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Abstract

The present application discloses a method, kit and application for detecting gene polymorphism based on shared primer probes. In the method, the reporter group is labeled on the 5' ends of the wild-type ARMS primers and mutant ARMS primers designed in the same direction to prepare the probe-specific ARMS primers, which are used for gene polymorphism detection after screening, especially for Human CYP2C19 polymorphism detection. The method has simple design, low cost, higher detection sensitivity, stronger specificity, and more accurate and objective results.

Description

technical field [0001] This application relates to the field of gene detection, in particular to a PCR detection of gene polymorphism. [0002] technical background [0003] At present, the methods used to detect gene polymorphism mainly include the following: sequencing typing method, high resolution melting curve typing method, gene chip typing method, TaqMan probe typing method and MGB probe typing method, etc. . Sequencing typing is the gold standard for detection in this field, but it has not been widely promoted due to the cumbersome operation and special equipment. The other methods are derived from PCR amplification technology and are relatively widely used. Among them, the detection process of sequencing typing method includes PCR amplification and sequencing, and the operation is cumbersome. The detection time is generally as long as 4 to 5 hours, and the PCR reaction tube needs to be opened during the operation process, which is easy to cause contamination of PCR ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/686C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/137C12Q2561/101C12Q2563/107
Inventor 扶媛媛周洋苏正稳曹彦东王利郝俊杨颖
Owner ANNGEEN BIOTECHNOLOGY CO LTD
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