Puenpa Jiratchaya, Suwannakarn Kamol, Chansaenroj Jira, Vongpunsawad Sompong, Poovorawan Yong
Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
J Virol Methods. 2017 Oct;248:92-99. doi: 10.1016/j.jviromet.2017.06.013. Epub 2017 Jun 27.
Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks.
用于检测肠道病毒71型（EV-A71）和柯萨奇病毒A16型（CV-A16）的实时逆转录聚合酶链反应（rRT-PCR）有助于快速准确地鉴定手足口病（HFMD）最常见的两种病原体。然而，HFMD中CV-A6感染在全球范围内的出现使得开发一种改进的多重rRT-PCR方法成为必要。为了快速确定HFMD的病因，开发了两种使用TaqMan探针的rRT-PCR检测方法，以区分三种选定的常见肠道病毒（EV-A71、CV-A16和CV-A6），并实现对肠道病毒的广泛检测（泛肠道病毒检测）。在所检测的其他RNA病毒中未观察到交叉反应。两种检测方法对EV-A71、CV-A6、CV-A16和泛肠道病毒的检测限均为每微升10拷贝。在检测2014 - 2016年期间的临床标本（n = 1049）时，这些方法显示出高准确性（EV-A71为90.6%；CV-A6为92.0%；CV-A16为100%）、敏感性（EV-A71为96.5%；CV-A6为95.8%；CV-A16为99.0%）和特异性（EV-A71为100%；CV-A6为99.9%；CV-A16为99.9%），优于传统RT-PCR。总体而言，多重rRT-PCR检测方法能够对EV-A71、CV-A6、CV-A16和肠道病毒进行高度灵敏的检测并快速同时分型，使其成为对与HFMD暴发相关的肠道病毒进行大规模监测的可行且有吸引力的方法。
