Two reverse-transcription-loop-mediated isothermal amplification systems for highly sensitive detection of enterovirus 71 and coxsackievirus A16

Abstract

Human enterovirus 71 and coxsackievirus A16 are two of the major causative agents of hand, foot, and mouth disease (HFMD). EV71 and CoxA16 infections in HFMD are clinically indistinguishable, but EV71 infection is more frequently associated with serious neurological complications and fatalities. In this study, two specific reverse-transcription-loop-mediated isothermal amplii¬cation (RT-LAMP) assays were developed for the detection of EV71 and CoxA16. High specificity systems for the EV71 and CoxA16 RT-LAMP systems have been confirmed by ruling out cross-reactivity with RNA of other common enteric viruses. The EV71 and CoxA16 RT-LAMP assays were found to be 10-fold and 100-fold more sensitive than conventional PCR, respectively, with detection limits of both 101 copies. To evaluate the application of the two RT-LAMP assays for clinical diagnosis, 60 stool specimens from suspect HFMD patients were detected and all samples were achieved in less than 60 min (even as little as 10 to 15 min). The positive rate of the EV71 RT-LAMP assay was higher (31/60=51.7%) than the RT-PCR assay (27/60=45%), while the positive rate of the CoxA16 RT-LAMP assay was equivalent to that of the RT-PCR assay (7/60=11.7%). These results indicate that the two RT-LAMP systems are potentially useful for rapid and sensitive diagnosis during HFMD outbreaks.   Key words: Enterovirus 71, coxsackievirus A16, reverse-transcription-loop-mediated isothermal amplification, hand foot, mouth disease.