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检测GI和GII族人源诺如病毒双重RT-qPCR的优化设计
Danlei Liu, Zilei Zhang, Qingping Wu, Peng Tian, Haoran Geng, Ting Xu, Dapeng Wang
工程（英文） ›› 2020, Vol. 6 ›› Issue (4) : 442-448.
PDF(1252 KB)
PDF(1252 KB)
检测GI和GII族人源诺如病毒双重RT-qPCR的优化设计
Redesigned Duplex RT-qPCR for the Detection of GI and GII Human Noroviruses
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。
Human noroviruses (HuNoVs) are major foodborne pathogens that cause nonbacterial acute gastroenteritis worldwide. As the tissue-culture system for HuNoVs is not mature enough for routine detection of the virus, detection is mainly dependent on molecular approaches such as reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The widely used primers and probes for RT-qPCR were established in the early 2000s. As HuNoVs are highly variant viruses, viral genome mutations result in previously designed primers and/or probes that were perfectly matched working less efficiently over time. In this study, a new duplex RT-qPCR (ND-RT-qPCR) was designed for the detection of genogroup I (GI) and genogroup II (GII) HuNoVs based on an analysis of viral sequences added in the database after 2010. Using long transcribed viral RNAs, the results demonstrate that the sensitivity of ND-RT-qPCR is as low as one genomic copy for both GI and GII HuNoVs. The performance of ND-RT-qPCR was further evaluated by a comparison with the commonly used Kageyama primer-probe sets for RT-qPCR (Kageyama RT-qPCR) for 23 HuNoV-positive clinical samples. All five GI samples were registered as positive by ND-RT-qPCR, whereas only two samples were registered as positive by Kageyama RT-qPCR. All 18 GII samples were registered as positive by ND-RT-qPCR, while 17 samples were registered as positive by Kageyama RT-qPCR. The sensitivity reflected by the Cq value was lower in ND-RT-qPCR than in Kageyama RT-qPCR. Our data suggest that ND-RT-qPCR could be a good fit for the detection of current strains of HuNoVs.
人源诺如病毒 / RT-qPCR / 优化设计 / 引物 / 探针 / 检测
Human noroviruses / RT-qPCR / Redesign / Primer / Probe / Detection
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