Poster: Diseases of Plants: Disease Detection & Diagnosis
Multiplex RT-PCR detection of eight viruses infecting cucurbits and discrimination using High Resolution Melting analysis
L. PEÑA ZUNIGA (1), F. Ochoa-Corona (1), A. Ali (2) (1) Oklahoma State University, U.S.A.; (2) University of Tulsa, U.S.A.
Cucurbits are one of the largest horticultural commodities in United States and Oklahoma largely contributes with watermelon production. However, the cucurbit cultivated acreage of Oklahoma had decreased, in part, due to viral infections. This research targets eight disease-causing viruses: Cucumber mosaic virus (CMVI and CMVII), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus (ZYMV). A rapid detection and discrimination method is needed for biosecurity and breeding for resistance against these viruses. Sequences from the polymerase protein gene (RNA) from the six genera were retrieved from NCBI and aligned using MEGA 6. Primer sets were designed from conserved regions using the web application PrimerQuest® and the thermodynamic features of each primer were tested using Oligonalyzer 3.1. A Multiplex RT-PCR was developed to be coupled to High Resolution Melting (HRM) analysis for rapid detection of the targeted viruses. The obtained melting profiles allowed a clear discrimination among these viruses. This Multiplex RT-PCR High Resolution Melting method has potential to be applied in epidemiological studies of viral diseases in cucurbit crops, microbial forensics and biosecurity.