Using of Duplex RT-PCR for Detection of Piper Yellow Mottle Virus and Cucumber Mosaic Virus, Infecting Black Pepper (Piper nigrum L.)

Abstract

Piper yellow mottle virus (PYMoV) and Cucumber mosaic virus subgroup I (CMV subgroup I) are important viruses that can cause significant loss of black pepper product around the world. The objective of this research aims to use a duplex reverse transcription-PCR (duplex RT-PCR) for the detection of PYMoV and CMV subgroup I infecting black pepper. Primer pairs specific to PYMoV (PYMoV-F and PYMoV-R) and CMV subgroup I (CMV I-F and CMV I-R) were designed from Open reading frame I (ORFI) of PYMoV and CP gene of CMV subgroup I which yields DNA products of approximately 450 bp and 500 bp, respectively. Optimization conditions for the duplex RT-PCR to amplify DNA products of the corresponding viruses were studied. The result showed that the optimized concentrations of the total DNA for PYMoV were 10, 50 and 100 ng whereas those for CMV subgroup I were 100 and 200 ng. The optimized annealing temperature was 55.8°C and the quantitative ratios of primer pairs for both viruses (PYMoV-F and PMMoV-R : CMV I-F and CMV I-R) were 1 : 1. Evaluation of the developed duplex RT-PCR was performed by analyzing nine black pepper leaf samples showing virus like-diseases. The result showed a strong DNA band at approximately 450 bp in all samples indicating that they were infected by PYMoV only. This result was correlated with those obtained from the conventional PCR and RT-PCR detections, respectively. Therefore, the developed duplex RT-PCR can be used for the detection of these two viruses. In addition, this technique will be useful in further screening application for virus-free black pepper production.