Application of reverse transcription and droplet digital PCR (RT-ddPCR) for detection of tomato torrado virus (ToTV) in greenhouse whitefly

Abstract

The technology of droplet digital PCR is considered to be third-generation technology of nucleic acid amplification recently developed for the diagnostics of phytopathogens, such as bacteria, nematodes, fungi and viruses. Due to its high sensitivity, precision and no need to use standard curves and/or reference genes it is an alternative to a real-time polymerase chain reaction (RT-qPCR). This paper presents the possibility of using the reverse transcription technique followed by ddPCR (RT-ddPCR) to detect tomato torrado virus, ToTV in the greenhouse whitefly (Trialeurodes vaporariorum), which is a real source of ToTV in tomato cultivation, especially in southern European countries, where it’s increased occurrence has been observed. The analysis of cDNA template evaluation of the virus RNA copies in a single adult greenhouse whitefly showed that it could carry from 220 to even 8600 copies of the virus gene. Due to the lack of effective antiviral agents, innovative and rapid viruses diagnostic assays are the essence of the fight against development of viral diseases. Therfore reported here RT-ddPCR protocol for the detection of ToTV in whiteflies may be an effective tool for monitoring the real threat of the tomato disease described by Spanish farmers as ”torrao”.