Poster: Diseases of Plants: Disease Detection & Diagnosis
Sensitive detection and discrimination of HPWMoV, WSMV and TriMV using multiplex RT-PCR and High Resolution Melting: Part II
A. LARREA-SARMIENTO (1), M. Arif (2), F. Ochoa-Corona (3), A. Olmedo-Velarde (4), J. Olson (3) (1) Universidad de las Américas, Ecuador; (2) Kansas State University, U.S.A.; (3) Oklahoma State University, U.S.A.; (4) Universidad de las Fuerzas Armadas ESP
High Plains wheat mosaic virus (HPWMoV), Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) cause disease in wheat crops in Central and Western USA, including Oklahoma and Kansas. Although these viruses are from different families, they are transmitted by Aceria tosichella, the wheat curl mite (Prostigmata: Eriophyidae). These viruses may infect simultaneously single plants. Melting curve analysis of RT-PCR products obtained by High Resolution Melting (HRM) enables to detect and to discriminate different virus species. The method is rapid, sensitive and allows discrimination among multiple samples. Specific primers for original single endpoint RT-PCR and reference positive controls from Agdia® were used as previously reported (Part I this research, 2013) to generate cDNA. uMELTSM analysis of expected PCR products was used to predict HRM scenarios. Special attention was given to PCR product size, GC percentage and melting temperature (Tm). Tm derived from dF/dT plots in vitro were similar to predictions by uMELTSM in silico. All primers sets showed a sensitivity down to 1 fg/uL from cDNA in single-endpoint RT-PCR reactions. HRM assays are sensitive and allows detection and discrimination of viruses causing diseases and significant economic losses in wheat and corn.