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用于检测和定量诺如病毒I型和II型基因组的实时逆转录聚合酶链反应检测方法的评估

Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

作者信息

Rupprom Kitwadee, Chavalitshewinkoon-Petmitr Porntip, Diraphat Pornphan, Kittigul Leera

机构信息

Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, 10400, Thailand.

Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.

出版信息

Virol Sin. 2017 Apr;32(2):139-146. doi: 10.1007/s12250-016-3863-9. Epub 2017 Feb 20.

Abstract

Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10 DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

摘要

诺如病毒是人类急性胃肠炎的主要病因。实时逆转录聚合酶链反应(实时RT-PCR)是一种很有前景的检测诺如病毒的分子方法。在本研究中,评估了三种TaqMan实时RT-PCR检测方法的性能,其中一种是市售的实时RT-PCR试剂盒(检测方法A:诺如病毒实时RT-PCR试剂盒),另外两种是内部实时RT-PCR检测方法(检测方法B:LightCycler RNA Master Hybprobe和检测方法C:RealTime ready RNA Virus Master)。检测方法A和B对诺如病毒GI的敏感性高于检测方法C,而它们对GII DNA标准对照的敏感性相同(10个DNA拷贝/毫升)。检测方法B对两个基因组的效率最高。在GI和GII诺如病毒、轮状病毒、甲型肝炎病毒和脊髓灰质炎病毒之间未观察到交叉反应。这些检测方法在GI和GII诺如病毒阳性粪便样本中的检出率无显著差异。然而,检测方法B对GII的平均定量循环(Cq)值低于检测方法A和C,具有统计学意义(P值,0.000)。所有三种实时RT-PCR检测方法都能检测多种诺如病毒,包括GI.2、GII.2、GII.3、GII.4、GII.6、GII.12、GII.17和GII.21。本研究表明,由于检测方法B具有良好的分析灵敏度,并且在DNA标准对照和临床样本上扩增诺如病毒的性能更高,因此它是检测和定量诺如病毒GI和GII的合适检测方法。

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