Rapid Detection and Differentiation of Three Cucurbit-infecting Poleroviruses by Multiplex RT–PCR

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) method was developed and optimized for the simultaneous detection and differentiation of three poleroviruses infecting cucurbits. Cucurbit aphid-borne yellows virus (CABYV), Melon aphid-borne yellows virus (MABYV) and Suakwa aphid-borne yellows virus (SABYV) could be differentiated simultaneously using four optimized specific oligonucleotide primers, including one universal primer for detecting poleroviruses and three virus-specific primers. Amplification of three target viruses was also optimized by increasing the PCR annealing temperatures. The mRT-PCR products consisted of fragments of 700 base pairs (bp) for CABYV, 450 bp for MABYV and 950 bp for SABYV. Detection limits of the RNA quantity for mRT-PCR were 1 pg for CABYV, 0.1 pg for MABYV and 1 pg for SABYV. No specific products could be amplified from RNA of other non-target poleroviruses. The mRT-PCR was found to be a specific, sensitive and cost-effective method for detecting multiple poleroviruses in cucurbits.