Detection of human Enterovirus 71 reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Abstract

In this study, a one-step, single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and validated for the detection of human Enterovirus 71 (EV71), the major aetiological agent of hand, foot and mouth disease (HFMD). Reverse transcription loop-mediated isothermal amplification assay was optimized to amplify VP1 gene in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for 1 h. Amplified products were evaluated by visual inspection and agarose gel electrophoresis. The detection limit of RT-LAMP assay was 10(-5) 100 TCID50 or 160 copies in samples after RNA extraction, which was 10-fold higher in sensitivity than traditional reverse transcription polymerase chain reaction (RT-PCR). The specific positive amplification was only observed in EV71 strains, while no amplification was detected in other tested viruses. Digestion with a specific Escherichia coli restriction enzymes V (EcoR V) demonstrated that the amplified product was unique. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of 33 clinical samples. Reverse transcription loop-mediated isothermal amplification is a novel, alternative microbiological approach for rapid, sensitive and specific detection of EV71 in HFMD. Reverse transcription loop-mediated isothermal amplification assay is suitable for the diagnosis of EV71 infection as a routine diagnostic tool for HFMD because of fewer requirements of experimental conditions such as private clinics, field applications as well as an epidemiological survey in epidemic areas. RT-LAMP can also be used as an alternative method for EV71 detection.