National Institute for Viral Disease Control and Prevention, China CDC, Beijing, PR China.
PLoS One. 2013 Apr 18;8(4):e61451. doi: 10.1371/journal.pone.0061451. Print 2013.
Large-scale Hand, Foot, and Mouth Disease (HFMD) outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs). Among them, human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1-2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells). The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11) by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.
自 2008 年以来，中国频繁发生大规模手足口病 (HFMD) 疫情，每年影响超过 100 万名儿童，导致数百名儿童死亡。HFMD 的病原体主要是人类肠道病毒 (HEVs)。其中，肠道病毒 71 型 (HEV71) 和柯萨奇病毒 A16 型 (CVA16) 是 HFMD 的最常见病原体。然而，其他 HEVs 也可能导致 HFMD。为了快速检测 HEV71 和 CVA16，并确保检测到所有引起 HFMD 的 HEVs，本研究开发了两种基于实时杂交探针的 RT-PCR 检测方法。一种是多重实时 RT-PCR 检测方法，旨在从临床标本中直接快速检测和区分 HEV71 和 CVA16，另一种是广谱实时 RT-PCR 检测方法，可检测几乎所有的 HEVs。实验证实，这两种检测方法具有很高的灵敏度和特异性，检测 HEVs、HEV71 和 CVA16 的灵敏度分别高达 0.1TCID50/ml。用三种方法，包括两种实时 RT-PCR 检测方法、直接常规 RT-PCR 检测方法和在人横纹肌肉瘤细胞 (RD 细胞) 上的病毒分离检测方法，同时检测了 213 份临床标本。实时 RT-PCR 检测 HEV71 和 CVA16 的总阳性率分别为 69.48%、47.42%和 34.58%。实时 RT-PCR 检测方法检测到的 1 份 HFMD 临床标本对 HEV 呈阳性，但对 HEV71 或 CVA16 呈阴性，经病毒分离、RT-PCR 和 VP1 基因测序鉴定为肠病毒 11 型 (Echo11)。这两种实时 RT-PCR 检测方法已应用于 31 个省级 HFMD 实验室，用于检测 HFMD 的病原体，有助于在 HFMD 疫情早期快速鉴定病原体，并有助于阐明中国 HFMD 的病原体。
