Reverse transcription loop-mediated isothermal amplification (RT-LAMP) designed for fast and sensitive on-site detection of Pepper chat fruit viroid (PCFVd)

Pepper chat fruit viroid (PCFVd) is one of the most important tomato and pepper diseases causing serious losses, affecting productivity, fruit quality and even international seed trade. Reverse transcription Loop-mediated isothermal amplification (RT-LAMP) is a fast and reliable RNA amplification assay, out competing conventional reverse transcription polymerase chain reaction (RT-PCR) in robustness, analytical sensitivity and specificity, and cost-effectiveness. In this work, a PCFVd specific RT-LAMP detection assay, based on a set of six primers was developed. Under the optimized conditions, PCFVd could be detected within 15 min, with a sensitivity of detecting PCFVd that was almost the same as a probe-based qRT-PCR and 10-100 times higher than the available RT-PCR methods. No cross-amplification with other viroids and tomato viruses was observed. The validated assay was also adapted for convenient on-site detection. Besides replacing the full RNA extraction with a simple lysis procedure, several visualization options, of which the use of SYTO9 was the most convenient, were presented to accommodate any in-field application of the method.