Children's Hospital Affiliated to Zhengzhou University, Henan International Joint Laboratory of Children's Infectious Diseases, China; Zhengzhou University, China.
Zhengzhou University, China.
J Virol Methods. 2024 Sep;329:114983. doi: 10.1016/j.jviromet.2024.114983. Epub 2024 Jun 18.
Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5'UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/μl, for the 5' UTR was 50.1187 copies/μl and for the RNaseP gene was 158.49 copies/μl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100 % (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94 % (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95 % (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5 hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.
手足口病（HFMD）是一种常见的儿童传染病，由人肠道病毒引起。本研究旨在开发一种快速、灵敏、准确的检测柯萨奇病毒 A16（CV-A16）引起的重症 HFMD 的方法。采用闭管敏感一步法逆转录定量实时聚合酶链反应（RT-qPCR）检测重症 HFMD 早期的 CV-A16。该检测方法针对 CV-A16 结构蛋白 VP1，用于区分 CV-A16 与其他柯萨奇病毒，肠道病毒的 5'UTR 区域用于检测肠道病毒，采用核糖核酸酶 P（RNaseP）作为内参基因。多重 MGB 探针检测系统用于在同一系统中检测具有不同荧光报告物的 PCR 扩增子。RT-qPCR 检测 CV-A16 VP1 基因的检测限（LOD）为 125.893 拷贝/μl，5'UTR 为 50.1187 拷贝/μl，RNaseP 基因为 158.49 拷贝/μl。此外，特异性分析表明，多重 RT-PCR 与流感病毒、疱疹性咽峡炎病毒和 SARS-COV-2 无交叉反应。在相关性分析中，多重 RT-qPCR 检测 CV-A16 的灵敏度为 100%（288/288），特异性为 99.94%（3395/3397）。多重 RT-qPCR 与临床诊断结果的总符合率为 99.95%（3683/3685），kappa 值为 0.996（p<0.001）。从标本处理到报告结果，整个过程可在 1.5 小时内完成。本研究建立的一步法多重 RT-qPCR 检测 CV-A16 方法是一种快速、可靠的实验室诊断工具，可用于快速区分检测 CV-A16，特别是用于病毒载量低的早期重症 HFMD 患者。
