Specific detection of Malus- and Pyrus-infecting viroids by real-time reverse-transcription quantitative PCR assays

Viroids present a number of issues for their detection and diagnosis because of the absence of symptom expression in many hosts and their low titers in infected plants. However, quarantine programs rely on symptom observations and routine diagnostic testing to reduce the risk of spreading viroid-infected materials to situations where they might affect crop health and production. Sensitive, accurate, and specific assays for viroid detection from both asymptomatic and symptomatic hosts are necessary for managing viroids in post-entry quarantine and certification schemes. The aim of this study was to develop and optimize superior assays based on the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for the specific detection of apple hammerhead viroid (AHVd), apple scar skin viroid (ASSVd) and pear blister canker viroid (PBCVd). The real-time RT-qPCR assays thus developed detected a greater range of viroid isolates and with greater sensitivity than the current endpoint RT-PCR assays, down to 10¹ copies per reaction without any amplification of the non-target viroid or virus sequences tested.