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Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16.
Virology Journal ( IF 4.0 ) Pub Date : 2019-12-30 , DOI: 10.1186/s12985-019-1264-z
Xin-Na Li 1 , Xin-Xin Shen 1 , Ming-Hui Li 2 , Ju-Ju Qi 1 , Rui-Huan Wang 1 , Qing-Xia Duan 1 , Rui-Qing Zhang 1 , Tao Fan 1 , Xue-Ding Bai 1 , Guo-Hao Fan 1 , Yao Xie 2 , Xue-Jun Ma 1
Affiliation  

BACKGROUND Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. METHODS Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. RESULTS The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. CONCLUSIONS The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

中文翻译:

双工实时和侧向流动条逆转录重组酶辅助扩增试验在检测肠病毒71和柯萨奇病毒A16中的适用性。

背景技术肠病毒71(EV71)和柯萨奇病毒A16(CA16)是手足口病(HFMD)的两种主要病因。在资源有限的环境中,简单快速检测EV71和CA16至关重要。方法分别开发了结合竞争性内部扩增对照(IAC)的双重实时逆转录重组酶辅助扩增(RT-RAA)分析和结合侧流试纸(LFS)的可见RT-RAA分析检测EV71和CA16。使用便携式实时荧光检测器在42°C下30分钟内进行双工实时RT-RAA分析,而在培养箱内30分钟内在42°C下进行LFS RT-RAA分析。含有保守的VP1基因的重组质粒用于分析这两种方法的敏感性。来自怀疑疑似手足口病感染患者的445个临床标本被用于评估检测的性能。结果EV71和CA16的双重实时RT-RAA的检出限(LoD)分别为每个反应47份和38份。EV71和CA16的LFS RT-RAA的LoD均为每个反应91份。与其他肠病毒没有交叉反应。与逆转录定量PCR(RT-qPCR)相比,双重实时RT-RAA检测对EV71的临床诊断敏感性为92.3%,对CA16为99.0%,临床诊断特异性分别为99.7和100%。LFS RT-RAA分析的临床诊断敏感性对EV71为90.1%,对CA16为94.9%,临床诊断特异性分别为99.7和100%。
更新日期:2019-12-31
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