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Primer set, method and kit for detecting slco1b1 and apoe gene polymorphisms based on shared primer probes

A gene polymorphism, APOE4 technology, applied in the field of PCR detection of gene polymorphism, can solve the problems of increased adverse reactions of statins, increased blood concentration of statins, and reduced OATP1B1 uptake capacity, etc.

Active Publication Date: 2020-08-11
ANNGEEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The SLCO1B1 gene encodes the organic anion transporting polypeptide OATP1B1. The 521T>C (Val174Ala) polymorphism in exon 5 of the gene is the main genetic variation in the Asian population, with an allele frequency of 10-15%. The polymorphism of the SLCO1B1 gene Significantly reduce the uptake ability of OATP1B1 to its substrate, increase the plasma concentration of statins such as pravastatin, atorvastatin and rosuvastatin, etc., leading to a significant increase in the adverse reactions of statins

Method used

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  • Primer set, method and kit for detecting slco1b1 and apoe gene polymorphisms based on shared primer probes
  • Primer set, method and kit for detecting slco1b1 and apoe gene polymorphisms based on shared primer probes
  • Primer set, method and kit for detecting slco1b1 and apoe gene polymorphisms based on shared primer probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Example 1 Design optimization of primers, optimization screening of markers

[0150] 1. The design and screening of wild-type and mutant-type probe ARMS primers, the specific steps are as follows:

[0151] 1) Preliminarily design a series of ARMS primers for SLCO1B1*5, SLCO1B1*1b, APOE2, APOE4 wild-type and mutant sites, with a Tm value of 58-62°C, and the sequences are shown in Table 1:

[0152] Table 1. Preliminary design of SLCO1B1*5, SLCO1B1*1b, APOE2, APOE4 wild-type and mutant ARMS primers

[0153]

[0154]

[0155]

[0156]

[0157] 2) SLCO1B1*5 wild-type ARMS primers, downstream primers, and SYBR-premix buffer to prepare PCR reaction solutions, respectively detect 5000 copy / μl of SLCO1B1*5 wild-type plasmids and mutant plasmids; SLCO1B1*5 mutant ARMS primers, downstream primers , SYBR-premix buffer to prepare PCR reaction solution, respectively detect 5000copy / μl SLCO1B1*5 wild-type plasmid and mutant plasmid; SLCO1B1*1b wild-type ARMS primer, downs...

Embodiment 2

[0199] Example 2 Primer preparation and kit assembly

[0200] 1. Synthesis and fluorescent labeling of probe-based ARMS primers, quenching probes, and downstream primers in this application

[0201] The probing ARMS primers, quenching probes, downstream primers and markers screened were optimized according to Example 1, and primer synthesis and fluorescent labeling were carried out. SLCO1B1*5 mutant type probe ARMS primer 5′-end labeled TET, SLCO1B1*5 quencher probe 3′-end labeled BHQ2, SLCO1B1*5 downstream primer; SLCO1B1*1b wild-type probe ARMS primer 5′-end labeled ROX , SLCO1B1*1b mutant type probe ARMS primer 5′-end labeled CY5, SLCO1B1*1b quencher probe 3′-end labeled BHQ2, SLCO1B1*1b downstream primer; APOE2 wild-type probe-type ARMS primer 5′-end labeled FAM, APOE2 mutant type probe ARMS primer 5′-end labeled TET, APOE2 quencher probe 3′-end labeled BHQ2, APOE2 downstream primer; APOE4 wild-type probe ARMS primer 5′-end labeled ROX, APOE4 mutant type probe ARMS The 5...

Embodiment 3

[0251] Embodiment 3 detection sensitivity

[0252] Use the kit a based on shared primer probes to detect human SLCO1B1 and APOE gene polymorphisms prepared in Example 2 and the detection kit b based on ARMS primers+TaqMan probes to detect SLCO1B1 and APOE gene polymorphisms, based on MGB probes Detect SLCO1B1 and APOE gene polymorphism kit c comparison test, the specific comparison test steps are as follows:

[0253] Step 1. Prepare sensitivity templates, including SLCO1B1*5 homozygous wild type 2ng / μl, 1ng / μl, 0.5ng / μl, 0.2ng / μl, SLCO1B1*5 homozygous mutant 2ng / μl, 1ng / μl, 0.5ng / μl, 0.2ng / μl, SLCO1B1*5 heterozygous 2ng / μl, 1ng / μl, 0.5ng / μl, 0.2ng / μl; SLCO1B1*1b homozygous wild type 2ng / μl, 1ng / μl, 0.5ng / μl μl, 0.2ng / μl, SLCO1B1*1b homozygous mutant 2ng / μl, 1ng / μl, 0.5ng / μl, 0.2ng / μl, SLCO1B1*1b heterozygous 2ng / μl, 1ng / μl, 0.5ng / μl , 0.2ng / μl; APOE2 homozygous wild type 2ng / μl, 1ng / μl, 0.5ng / μl, 0.2ng / μl, APOE2 homozygous mutant 2ng / μl, 1ng / μl, 0.5ng / μl, 0.2ng / μl μl, APOE...

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Abstract

The invention discloses a primer group, a method and a kit for detecting SLCO1B1 and APOE gene polymorphism based on a shared primer probe. According to the method, reporter groups are marked at 5'ends of wild ARMS primers and mutant ARMS primers which are designed in the same direction to prepare ARMS primers with probes, and the ARMS primers are used for polymorphism detection of genes after being screened. The method is higher in detection sensitivity, stronger in specificity, more accurate and objective in result, simple in design and low in cost.

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): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2535/137C12Q2563/107C12Q2531/113C12Q2545/113
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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