Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification assays for Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 in Clinical Samples

Hong Zhang, Kai Nie, Yunzhi Liu, Le Luo, Wei Huang, Shuaifeng Zhou, Mengjie Yang, Yu Chen, Jianmin Luo, Lidong Gao, Xuejun Ma

Department of Microbiology, Center for Disease Control and Prevention of HuNan, Changsha, China.
State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.

DOI: 10.4236/aid.2012.24019                

Abstract

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.

Keywords

Human Enterovirus 71; Coxsackievirus A16; Reverse Transcription Loop-Mediated Isothermal Amplification

Conflicts of Interest

The authors declare no conflicts of interest.

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