Guan Li, Xu Songtao, Nie Kai, Zhang Dan, Li Xinna, Xu Wenbo, Ma Xuejun
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
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Zhonghua Yu Fang Yi Xue Za Zhi. 2015 Nov;49(11):1009-13.
To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).
The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease.
A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%.
The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.
基于羟基萘酚蓝（HNB）的颜色变化，开发一种简单、快速且灵敏的比色逆转录环介导等温扩增（RT-LAMP）方法，用于快速检测柯萨奇病毒A6（CV-A6）。
该方法采用一组六条引物，识别VP1基因序列，在63℃等温条件下扩增核酸50分钟。通过预先添加HNB染料，目视检查颜色变化来检测产物。通过检测一系列不同的人肠道病毒来验证特异性。通过对CV-A6 VP1基因体外转录的RNA分子进行系列稀释来评估该检测方法的灵敏度，并与实时逆转录聚合酶链反应（rRT-PCR）进行平行比较。用92份手足口病患者的临床标本对该检测方法进行评估。
仅在CV-A6制备物中观察到阳性颜色（天蓝色），而其他23种人肠道病毒均未出现颜色变化。基于HNB的RT-LAMP检测灵敏度为100拷贝/反应，与rRT-PCR处于同一水平。RT-LAMP对92份临床标本分析的结果与rRT-PCR一致。两种方法的kappa相关性为1，RT-LAMP检测的灵敏度和特异性均为100%。
所建立的RT-LAMP检测方法具有良好的特异性和灵敏度，因此被证明是一种用于CV-A6感染的有前景的筛查工具。它也有潜力用于资源有限的临床场所和现场研究。
