Improvement in the sensitivity of viroid detection by adapting the reverse transcription step in one-step RT-qPCR assays

Abstract

Over the course of developing and applying a new real-time PCR assay for the detection of the newly described apple chlorotic fruit spot viroid (ACFSVd), slight modifications of the reverse transcription (RT) step were found to improve significantly the detection limit of the assay. To prove this hypothesis, three different one-step RT-qPCR kits for the detection of three plant viroids and three plant viruses were compared. The results showed both extension of the RT reaction time from 10 or 15 min-30 min or the increase in reaction temperature from 49 to 52 °C for the cDNA synthesis step results in a 10 times higher sensitivity for potato spindle tuber viroid and apple scar skin viroid one-step RT-qPCR assay and 45 higher sensitivity for ACFSVd one-step RT-qPCR assay. No variation in the detection limit was observed when the modifications were tested on tomato brown rugose fruit virus, plum pox virus and tomato ringspot virus assays. This finding is highly valuable for the investigation of viroids in general and could contribute to enhance sensitivity in their detection and to benefit regulatory outcomes for national plant protection organisations.

Keywords: ACFSVd; Detection limit; Improvement; One-step RT-qPCR; Reverse transcriptase.