双重实时和侧流条逆转录重组酶辅助扩增检测法在检测肠道病毒 71 型和柯萨奇病毒 A16 中的适用性。
Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16.
机构信息
NHC Key Laboratory of Medical Virology and Viral Diseases, Chinese Center for Disease Control and Prevention, National Institute for Viral Disease Control and Prevention, No.155 Changbai Road, Changping district, Beijing, 102206, China.
Department of hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Jing Shun Dong Jie 8#, Beijing, 100015, China.
出版信息
Virol J. 2019 Dec 30;16(1):166. doi: 10.1186/s12985-019-1264-z.
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.
背景
肠道病毒 71 型（EV71）和柯萨奇病毒 A16 型（CA16）是手足口病（HFMD）的两个主要病原体。在资源有限的环境中，快速简便地检测 EV71 和 CA16 至关重要。
方法
分别开发了包含竞争性内部扩增对照（IAC）的双实时 RT-RAA 检测和带有侧流条（LFS）的可视 RT-RAA 检测，用于检测 EV71 和 CA16。双实时 RT-RAA 检测可在 42°C 下于 30 分钟内使用便携式实时荧光检测仪完成，而 LFS RT-RAA 检测可在 42°C 下于 30 分钟内在孵育器中完成。使用包含保守 VP1 基因的重组质粒来分析这两种方法的灵敏度。共使用 445 份来自疑似 HFMD 感染患者的临床标本来评估这些检测方法的性能。
结果
双实时 RT-RAA 检测 EV71 和 CA16 的检出限（LoD）分别为 47 拷贝和 38 拷贝/反应。LFS RT-RAA 检测 EV71 和 CA16 的 LoD 均为 91 拷贝/反应。与其他肠道病毒无交叉反应。与逆转录定量 PCR（RT-qPCR）相比，双实时 RT-RAA 检测的临床诊断灵敏度分别为 EV71 92.3%和 CA16 99.0%，临床诊断特异性分别为 99.7%和 100%。LFS RT-RAA 检测的临床诊断灵敏度分别为 EV71 90.1%和 CA16 94.9%，临床诊断特异性分别为 99.7%和 100%。
结论
开发的用于检测 EV71 和 CA16 的双实时 RT-RAA 和 LFS RT-RAA 检测方法在初级临床环境中具有潜在的应用价值。
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