Development of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) that detects enteroviruses by targeting the highly conserved 5'-UTR region.

Abstract

Enteroviruses are positive sense single-stranded RNA viruses. Most infections from enteroviruses are asymptomatic and can circulate "silently", increasing the risk of an outbreak. For preventing such outbreaks, a rapid, cost-effective, and simple assay for the detection of enteroviruses is of great importance. In this study, we developed a rapid, simple, sensitive, and specific isothermal reverse transcription assay (RT-Loop-Mediated Amplification, RT-LAMP) for the detection of EV-A, B, C, and D species of enteroviruses, by targeting the highly conserved 5'UTR region. The assay was designed and validated based on reference sequences of the four species and on clinical and environmental isolates. The limit of detection of the assay is 0.75 CCID50/assay for Enterovirus A, B, and D and 0.075 CCID50/assay for Enterovirus C. LAMP allows immediate diagnosis in just 30-50min, instead of a minimum of 120min needed for PCR with an equal or better sensitivity. Moreover, due to its isothermal nature, there is no need for expensive equipment, thus decreasing the cost of each reaction. Therefore, this assay is ideal for use in resource-limited settings such as primary care facilities and environmental and clinical laboratories in developing countries.