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《工程(英文)》 >> 2020年 第6卷 第4期 doi: 10.1016/j.eng.2019.08.018

检测GI和GII族人源诺如病毒双重RT-qPCR的优化设计

a Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
b State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, China
c Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94706, USA

# These authors contributed equally to this work.

收稿日期: 2018-08-29 修回日期: 2018-11-19 录用日期: 2019-08-30 发布日期: 2020-03-03

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摘要

人源诺如病毒(human norovirus, HuNoV)是重要的食源性病毒之一,能够引起全世界范围人类的非细菌性急性胃肠炎。由于HuNoV的体外培养体系尚不成熟,无法用于常规的病毒分离和检测,因此目前对HuNoV的检测依赖于分子方法,如逆转录聚合酶链反应(reverse transcription polymerase
chain reaction, RT-PCR)和逆转录实时定量聚合酶链反应(reverse transcription quantitative real-time polymerase chain reaction, RT-qPCR)。21世纪初期设计的引物和探针依然被广泛地用于RTqPCR检测体系。HuNoV基因组具有变异度高的特点,导致设计的引物和(或)探针无法有效地与已进化的新病毒核酸进行匹配,从而随着时间的推移效率降低。为了提高HuNoV检出效率,本研究基于2010年后GenBank公布的病毒序列,分析设计了一套HuNoV检测引物和探针,并优化了一种新的双重RT-qPCR(new duplex RT-qPCR, ND-RT-qPCR)检测体系。以体外转录获得的长链病毒RNA为模板,ND-RT-qPCR可对低至一个基因组的GI和GII族HuNoV进行有效检测。以23份HuNoV临床样本为对象,评估了ND-RT-qPCR和常用RT-qPCR(Kageyama RT-qPCR)的检测性能。结果显示:ND-RT-qPCR检出所有GI族样本(5/5),而Kageyama RT-qPCR只检出两个样本(2/5)。ND-RT-qPCR检出18个GII族样本(18/18),而Kageyama RT-qPCR漏检1个样本。另外,ND-RTqPCR的灵敏度显著高于Kageyama RT-qPCR(前者Cq值低于后者Cq值)。因此,ND-RT-qPCR有助于提高HuNoV检出率,是目前该病毒检测良好的选择。

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