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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC ENHANCEMENT FOR RESISTANCE TO BIOTIC AND ABIOTIC STRESSES IN HARD WINTER WHEAT

Location: Hard Winter Wheat Genetics Research Unit

Title: Multiplex Real Time PCR For Detection of Wheat Streak Mosaic Virus and Triticum Mosaic Virus

Authors
item Price, Jacob -
item Smith, Jessica -
item Simmons, Angela -
item Fellers, John
item Rush, Charlie -

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 26, 2010
Publication Date: February 4, 2010
Citation: Price, J.A., Smith, J.T., Simmons, A., Fellers, J.P., Rush, C.M. 2010. Multiplex Real Time PCR For Detection of Wheat Streak Mosaic Virus and Triticum Mosaic Virus. Journal of Virological Methods. 165: 198-201

Interpretive Summary: Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are widespread throughout the southwestern Great Plains states. Both viruses have similar symptoms which makes visual identification almost impossible. The current ELISA method for detection of the viruses in plant samples uses antibodies specific to each of the viruses, but can be insensitive to low concentrations and depends on having high quality antiserum. The PCR method can be more sensitive and does not depend on antiserum. In both cases, only one of the viruses can be tested for per assay. The work that is being reported is a new multiplex method to test for both viruses at the same time in the same assay. Because of the higher sensitivity, it is superior to previous testing methods and can be used to more rapidly and more accurately diagnose these viruses.

Technical Abstract: Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TRIMV) are widespread throughout the southwestern Great Plains states. Using conventional diagnostics such as Enzyme-Linked Immunosorbent Assays (ELISA), these two viruses are commonly found together in infected wheat samples. Methods for molecular detection have been developed for wheat viral pathogens, but until recently no multiplex method for detection of both WSMV and TriMV within a single sample was available. Therefore, the objective of this study was to develop a multiplex real-time PCR technique for detection of both pathogens within a single plant sample. Specific primers and probe combinations were developed for detection of WSMV and TriMV, single and multiple reactions were run simultaneously to detect any loss in sensitivity during the multiplex reaction, as well as any cross reaction with other common wheat viruses. The multiplex reaction was successful in detection of both pathogens, with little difference between single and multiplex reactions, and no cross reaction was found with other common wheat viruses. This multiplex technique will be useful not only for diagnostic evaluations, but also as a valuable tool for ecological and epidemiology studies, and investigations of host/pathogen interactions, especially when the host is infected with both pathogens.

Last Modified: 7/28/2014
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