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

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.

Highlights

Human enteroviruses (HEVs) comprise more than 100 serotypes in four species (HEV-A to human enteroviruses (HEV)-D) in the genus Enterovirus, family Picornaviridae
In parallel with commercial quantitative real-time polymerase chain reaction diagnostic kits for enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), the reverse transcription loop-mediated isothermal amplification (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
Hand-Foot-andMouth Disease (HFMD) cases caused by human enterovirus 71(EV71) infections have been found to be associated with severe neurological complications at much higher rates than those caused by coxsackievirus A16 (CVA16) infections in Asia Pacific region [1,2]

Summary

Introduction

Human enteroviruses (HEVs) comprise more than 100 serotypes in four species (HEV-A to HEV-D) in the genus Enterovirus, family Picornaviridae. HFMD cases caused by human enterovirus 71(EV71) infections have been found to be associated with severe neurological complications at much higher rates than those caused by coxsackievirus A16 (CVA16) infections in Asia Pacific region [1,2]. A number of real-time RT-PCR assays for detection of EV71 and CVA16 with high specificities and sensitivities have been reported [1,7,8]. This method might not be suitable in primary clinical settings in developing countries or for field use because of the sophisticated instrumentation required and expensive reagents

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Results

Conclusion