诊断学理论与实践

诊断学理论与实践 ›› 2020, Vol. 19 ›› Issue (04): 402-406.doi: 10.16150/j.1671-2870.2020.04.015

• 论著 • 上一篇    下一篇

荧光聚合酶链反应检测血液标本快速方法的建立及其在ADRB1单核苷酸多态性分型中的应用

龚如涵, 李佳, 黄凯峰()   

  1. 上海交通大学附属第一人民医院检验医学科,上海 201620
  • 收稿日期:2019-11-18 出版日期:2020-08-25 发布日期:2022-07-15
  • 通讯作者: 黄凯峰 E-mail:89671801@qq.com

Establishment of fluorescence PCR method using blood specimens for a rapid genetyping and its application in ADRB1 genotyping

GONG Ruhan, LI Jia, HUANG Kaifeng()   

  1. Department of Clinical Laboratory, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 201620, China
  • Received:2019-11-18 Online:2020-08-25 Published:2022-07-15
  • Contact: HUANG Kaifeng E-mail:89671801@qq.com

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摘要：

目的： 建立一种荧光聚合酶链反应（polymerase chain reaction,PCR）检测血液标本的快速方法,并将其应用到ADRB1（1165G>C）基因的单核苷酸多态性（single nucleotide polymorphism,SNP）分型检测中。方法： 建立可直接利用血液样本进行荧光PCR扩增法,与传统血液样本的基因SNP检测方法不同,本所建立的方法无需进行血液基因组DNA的提取。选取1 051份临床乙二胺四乙酸（ethylene diamine tetraacetic acid,EDTA）抗凝全血样本,利用本研究建立的采用血液标本直接扩增的荧光PCR方法,进行ADRB1的SNP分型,并将分型结果与Sanger测序法这一“金标准”进行对比,评估两者之间的符合率和一致性。结果： 本研究建立的方法与传统DNA提取加荧光PCR方法相比,具有较好的扩增性能;与Sanger测序法相比,本研究建立的方法在ADRB1（1165G>C）基因SNP分型检测中的总体符合率达到98.98%,Kappa一致性系数达到0.971,具有较高的准确率。结论： 与传统方法相比,本研究建立的方法可省略基因组DNA提取步骤,其操作简便、成本较低,且准确率高,适合在基因分型相关临床检验工作中推广和使用。

关键词: 荧光聚合酶链反应, 直接扩增, ADRB1基因, Sanger测序

Abstract:

Objective: To establish a fluorescence PCR method using blood specimen for a rapid genetyping and apply it to the single nucleotide polymorphism(SNP) typing of ADRB1 (1165G>C) gene. Methods: The fluorescence PCR method using blood samples for gene amplification was established without extraction of blood genomic DNA, which was different from a traditional way. A total of 1 051 whole-blood samples anticoagulated with ethylene diamine tetraacetic acid (EDTA) were collected for ADRB1 (1165G>C) gene SNP typing. The performance of self-established method was assessed by comparison with results from Sanger sequencing method, and conformity and consistency (Kappa) between the two methods were calculated. Results: The method established in this research had better amplification performance. The overall typing coincidence rate of method established with Sanger sequencing reached 98.98%, and the Kappa consistency coefficient between them was 0.971. Conclusions: Compared with traditional methods, the method established in this research is easy to operate by omitting the step of genomic DNA extraction with low cost and high accuracy. It is suitable for popularization and application to clinical detection relate to genetyping.

Key words: Fluorescent polymerase chain reaction, Direct amplification, ADRB1 gene, Sanger sequencing

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