Optimization of multiplex PCR technique for the detection of viruses causing severe mosaic disease in pumpkin

Authors

  • Chinnaraja Chinnadurai Department of Life Sciences, Faculty of Science and Technology, The University of the West Indies
  • Adesh Ramsubhag Department of Life Sciences, Faculty of Science and Technology, The University of the West Indies
  • Jayaraj Jayaraman Department of Life Sciences, Faculty of Science and Technology, The University of the West Indies

Keywords:

ZYMV, SqMV, CMV, Pumpkin viruses, Multiplex RT-PCR

Abstract

Symptoms of severe mosaic and yellowing of leaves and severe deformation of pumpkin fruits have been noticed in field plants throughout Trinidad during recent years. Severe mosaic symptoms are normally associated with multiple virus infections which is nearly impossible to diagnose. In order to molecularly identify the viruses, RNAs were extracted from leaf samples collected from symptomatic plants taken from farmers? fields located at Barrackpore, Waterloo and Orange Grove. For developing multiplex PCR technique, RT-PCR was carried out with potyvirus, comovirus and cucumovirus specific primers. Strong amplifications were obtained for all the samples for Zucchini yellow mosaic virus (ZYMV) with ZYMVcp-for/ZYMVcp-rev primers targeting the 100bp of coat protein region. Seven out of ten samples showed amplification for Squash mosaic virus (SqMV) with SqMV RNA2-F1/SqMV RNA2-R1 primers targeting the 582bp of RNA-2 region. Similarly, four samples amplified for Cucumber mosaic virus (CMV) with CMV-F/CMV-R primers targeting the 530 bp coat protein region. Optimization was done for conducting multiplex PCR targeting all the three viruses in a single PCR reaction. The optimized multiplex RT-PCR was found to be equally efficient to uniplex-RT-PCR to amplify the target viruses from symptomatic plants. Use of multiplex PCR will have immense application in pumpkin disease monitoring, seed certification and quarantine programs

Issue

Section

Research Papers