Ding Xiong, Nie Kai, Shi Lei, Zhang Yong, Guan Li, Zhang Dan, Qi Shunxiang, Ma Xuejun
School of Light Industry and Food Sciences, South China University of Technology, Guangzhou city, Guangdong, China Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China.
Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, China.
J Clin Microbiol. 2014 Jun;52(6):1862-70. doi: 10.1128/JCM.03298-13. Epub 2014 Mar 19.
Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses.
快速检测人肠道病毒71型（EV71）和柯萨奇病毒A16型（CVA16）在手足口病（HFMD）早期阶段至关重要。在本研究中，我们开发并评估了一种新型逆转录等温多重自匹配引发扩增（RT - IMSA）检测方法，该方法利用逆转录酶和链置换DNA聚合酶快速检测EV71和CVA16。建立了使用浊度仪的实时RT - IMSA检测方法以及用于检测EV71和CVA16的可视化RT - IMSA检测方法，且均在1小时内完成，并采用针对VP1基因相同区域的已报道的实时逆转录环介导等温扩增（RT - LAMP）检测方法作为平行试验。通过检测体外转录的VP1 RNA，实时RT - IMSA检测方法的定量线性更好，EV71的R²值为0.952，CVA16的R²值为0.967，而实时RT - LAMP检测方法中EV71的R²值为0.803，CVA16的R²值为0.904。此外，实时RT - IMSA检测方法的检测限（EV71约为937拷贝/反应，CVA16约为67拷贝/反应）高于实时RT - LAMP检测方法（EV71约为3266拷贝/反应，CVA16约为430拷贝/反应），可视化RT - IMSA检测方法也观察到类似结果。新方法对相应靶标也具有高特异性，未观察到交叉反应。在临床评估中，与商业逆转录定量PCR（qRT - PCR）试剂盒相比，实时RT - IMSA检测方法的诊断敏感性（EV71为96.4%，CVA16为94.6%）高于实时RT - LAMP检测方法（EV71为91.1%，CVA16为90.8%）。可视化RT - IMSA检测方法也呈现相同结果。总之，这项概念验证研究表明，新型RT - IMSA检测方法在检测限方面优于RT - LAMP检测方法，具有快速检测EV71和CVA16病毒的潜力。
