Development of a fast and sensitive on-site diagnosis, reverse transcription loop mediated isothermal amplification (RT-LAMP), for Pepper chat fruit viroid (PCFVd)

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and reliable RNA di-agnostic assay, outcompeting distinctly conventional reverse transcription polymerase chain reaction (RT-PCR) in robustness, analytical sensitivity, and specificity. In addition, it is also highly cost-effective, and the procedure is far less time consuming. In this work, a PCFVd (Pepper chat fruit viroid) specific RT-LAMP detection assay was developed, based on a set of six primers. PCFVd, one of the most im-portant tomato and pepper diseases in Thailand, can be detected within 15 minutes with a sensitivity that is comparable as a probe-based RT-qPCR and 10-100 times higher than the available endpoint RT-PCR methods. No cross-amplification with other viroids and tomato viruses was observed. The on-site diagnostic assay was adapted for on-site applications with a simple plant lysis procedure and SYTO-9, fluorescence dye, for visualization under both visible and UV lights. In the closed-tube reaction mix, positive reactions displayed color change from yellow to lime green under visible light and fluorescence under UV light. The results indicate that this PCFVd RT-LAMP assay will be useful for any in-field appli-cation of the method.