Colorimetric detection of coxsackievirus A6 by reverse transcription loop mediated isothermal amplification
10.3760/cma.j.issn.0253-9624.2015.11.016
- VernacularTitle:基于颜色判定的环介导逆转录等温扩增技术检测柯萨奇病毒A6型
- Author:
Li GUAN
1
;
Songtao XU
;
Kai NIE
;
Dan ZHANG
;
Xinna LI
;
Wenbo XU
;
Xuejun MA
Author Information
1. 中国疾病预防控制中心病毒病预防控制所
- Keywords:
Polymerase chain reaction;
Coxsackievirus infections;
Loop-mediated isothermal amplification
- From:
Chinese Journal of Preventive Medicine
2015;49(11):1011-1015
- CountryChina
- Language:Chinese
-
Abstract:
Objective 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).Methods The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 ℃ 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.Results 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％.Conclusion 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.
