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Title: Development of a multiplex real-time PCR assay for the simultaneous detection of three seedborne pathogen types in cucurbits

item Ling, Kai-Shu
item Wechter, William - Pat

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 4/30/2008
Publication Date: 7/26/2008
Citation: Ling, K., Wechter, W.P., Walcott, R.R., Keinath, A.P. 2008. Development of a multiplex real-time PCR assay for the simultaneous detection of three seedborne pathogen types in cucurbits. Phytopathology. 98(6 supplement):S91.

Interpretive Summary:

Technical Abstract: Cucurbits (e.g., watermelon, melon, cucumber, squash, and pumpkin) are important crops in the U.S. Cucurbit diseases incited by seed-borne pathogens, such as bacterial fruit blotch [Acidovorax avenae subsp. citrulli (AAC)], gummy stem blight [Didymella bryoniae (DB)], and squash mosaic [Squash mosaic virus (SqMV)] are especially difficult to control. Effective management of these diseases is often through the use of certified pathogen-free seed lots. Current individual seed health testing methods (e.g., seedling grow-out, culturing or ELISA) are cumbersome and expensive. The objective of this study was to develop a single molecular technique (e.g., real-time PCR) for the simultaneous detection of several pathogen types (viral, bacterial and fungal) in cucurbit seeds. Previously, we developed a real-time PCR system for two pathogens (AAC and DB). Thus, in this study, we focused our efforts in developing a real-time reverse-transcriptase-PCR system for SqMV detection and evaluating the optimum conditions in the multiplex system. To maximize the broad-spectrum detection of various SqMV isolates, a conserved genome sequence region was identified through alignment of multiple SqMV sequences in GenBank. Primers and probe were designed based on the consensus sequence and shown to yield a high signal value in the simplex system prior to multiplexing. Converting the viral RNA to cDNA stabilized the templates for subsequent PCR amplification and allowed for simultaneous detection of three pathogen types in a single reaction. The multiplex real-time PCR detection system was capable of detecting three different pathogens in mixtures of infested seeds.