New and fast detection of Little cherry virus 1 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Little cherry virus 1 (LChV-1, Velarivirus, Closteroviridae) is an economically important pathogen affect-ing mainly cherry around the world emphasizing the impetus for its efficient and accurate on-site de-tection. This study describes the development of a reliable diagnostic protocol of LChV-1 based on a fast, sensitive, and easy-to-use one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The protocol detects all LChV-1 isolates in less than 10 min by fluorescence monitoring using a mobile detection device (GENIE, OptiGene) and is most optimal when incubation is performed at 67 °C. Sharp melting curves and unique melting temperatures (Tm) were obtained for the positive samples. Both the LAMP and classical RT-PCR methods are capable of specifically detecting LChV-1 in infected leaf tissues, but this validated leaf-to-result assay has remarkable advantages in comparison to RT-PCR. It is at least hundred fold more sensitive, significantly faster, and efficient at minimal cost, and is ready for on-field applications. In conclusion, this innovative LAMP approach can contribute to implementation of sustainable integrated management strategies for on-site detection of LChV-1 in commercial orchards or for horticultural research stations. It is also suitable for decision support in phytosanitary epidemiological programs.

Jaar van publicatie:2018