Development of real-time PCR based assays for simultaneous and improved detection of citrus viruses

Citrus, one of the most economically important crops, is susceptible to a number of arthropod- and graft-transmissible pathogens. Rapid and reliable methods for detecting multiple pathogens are important for routine diagnosis by reducing time, labour and costs. To this end, primers and TaqMan probes for Citrus psorosis virus (CPsV) and Citrus variegation virus (CVV) detection by singleplex realtime (q) reverse transcription (RT)- PCR were initially designed. Further optimizations included the development of a multiplex (m) RT-qPCR assay to detect simultaneously CPsV, CVV, and Citrus tristeza virus (CTV) in a single reaction. When 10-fold serial dilutions prepared using total RNAs from CPsV- and CVV-infected plants were tested, RT-qPCR assays proved to be 100 and 1000 times more sensitive than conventional RT-PCR, respectively. The target viruses were effectively identified by mRT-qPCR in fieldinfected clementine and sweet orange trees. The optimized multiplex assay proved to be as sensitive as the singleplex tests, thus providing a valuable alternative tool for detection of these citrus viruses.