一种通用型肠道病毒微滴式数字PCR检测方法的建立及应用

doi:10.13217/j.scjpm.2019.0015

华南预防医学 ›› 2019, Vol. 45 ›› Issue (1): 15-20.doi: 10.13217/j.scjpm.2019.0015

一种通用型肠道病毒微滴式数字PCR检测方法的建立及应用

袁润余¹, 曾汉日¹, 苏娟¹, 黎薇¹, 莫艳玲¹, 陆靖², 柯昌文¹

1.广东省疾病预防控制中心,广东广州 511430;
2.广东省疾病预防控制中心广东省公共卫生研究院

收稿日期:2018-08-20 发布日期:2019-04-19
作者简介:袁润余（1985—）,女,博士研究生,副研究员,主要从事人兽共患传染病的研究工作
基金资助:
国家科技重大专项（2018ZX09739002-004）; 广东省医学科学基金（A2018187）; 中山市经济和信息化局合作项目（中经信函【2013】634号）

Establishment and application of universal droplet digital polymerase chain reaction method for quantification of enterovirus

YUAN Run-yu¹, ZENG Han-ri¹, SU Juan¹, LI Wei¹, MO Yan-ling¹, LU Jing², KE Chang-wen¹

1.Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 511430, China;
2.Guangdong Provincial Institute of Public Health,Guangdong Provincial Center for Disease Control and Prevention

Received:2018-08-20 Published:2019-04-19

摘要/Abstract

摘要： 目的建立一种肠道病毒通用型微滴式数字PCR（ddPCR）的定量检测方法,以实现肠道病毒的量化检测。方法利用肠道病毒标准品对ddPCR反应中的探针浓度、退火温度进行优化,并确定ddPCR的检测范围。利用已优化好的反应条件对28份临床样本进行病毒载量检测。结果本研究确定ddPCR的最佳探针浓度为0.4 μmol/L,退火温度为51.0 ℃,核酸检测范围为3.02~3.59×10⁶ copies/μL,检出限为3.02 copies/μL。ddPCR方法线性相关系数为0.993 8,呈良好的线性关系。结论本研究建立基于ddPCR肠道通用型的定量方法,可有效地对临床样本中不同血清型的肠道病毒临床样本进行拷贝数定量分析,为临床研究病毒载量的测定提供一种技术。

关键词: 数字PCR, 肠道病毒属, 实时荧光定量PCR

Abstract: Objective To establish a quantitative detection method of droplet digital polymerase chain reaction （ddPCR） for identification and quantification of enterovirus. Methods The probe concentration and annealing temperature in ddPCR were optimized using enterovirus standards, and the detection range of ddPCR was determined. Viral load assays were performed on 28 clinical samples using optimized reaction conditions. Results In this study, the optimal probe concentration for ddPCR was 0.4 μmol/L, the annealing temperature was 51 °C, the nucleic acid detection range was 3.02-3.59×10⁶ copies/μL, and the detection limit was 3.02 copies/μL. The linear correlation coefficient of the ddPCR method was 0.993 8, showing a good linear relationship. Conclusion This study established a quantification method based on ddPCR for general enterovirus, which can effectively quantify enterovirus load in clinical samples and provide a technique for the determination of viral load in clinical research.

Key words: Digital PCR, Enterovirus, Real-time fluorescence quantitative PCR

中图分类号:

R117

袁润余, 曾汉日, 苏娟, 黎薇, 莫艳玲, 陆靖, 柯昌文. 一种通用型肠道病毒微滴式数字PCR检测方法的建立及应用[J]. 华南预防医学, 2019, 45(1): 15-20.

YUAN Run-yu, ZENG Han-ri, SU Juan, LI Wei, MO Yan-ling, LU Jing, KE Chang-wen. Establishment and application of universal droplet digital polymerase chain reaction method for quantification of enterovirus[J]. South China Journal of Preventive Medicine, 2019, 45(1): 15-20.

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一种通用型肠道病毒微滴式数字PCR检测方法的建立及应用

Establishment and application of universal droplet digital polymerase chain reaction method for quantification of enterovirus

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