Development of a Real-time RT-PCR Assay for Squash Mosaic Virus Useful for Broad Spectrum Detection of Various Serotypes and its Incorporation into a Multiplex Seed Health Assay

摘要

Seed-borne pathogens pose a serious threat to modern agricultural cropping systems, as they can be disseminated to many geographical regions around the world. With trends of increasing global seed production and trade, seed-health testing is an important quality control step to prevent the introduction of harmful pathogens into agricultural production systems. An effective seed-health assay depends on a test that can provide timely, sensitive and broad-spectrum detection of all genetic variants of a pathogen, or in some cases, of several different pathogens. Previously, we developed a real-time PCR (qPCR) assay that would permit the simultaneous detection of two major seed-borne pathogens of cucurbits, the bacterium Acidovorax avenae subsp. citrulli (AAC, the causal agent of bacterial fruit blotch) and a fungus Didymella bryoniae (DB, the causal agent of gummy stem blight). The objective of the present study was to develop a sensitive, reverse transcriptase (RT)-based, qRT-PCR for broad spectrum detection of both serotypes of Squash mosaic virus (SqMV), that could be incorporated into a simultaneous detection of three pathogen types in a single PCR reaction. Converting SqMV RNA to cDNA prior to multiplexing stabilized the viral template that was then mixed with two other DNA templates (AAC and DB). To facilitate seed health testing, a generic plant nucleic acid extraction method was developed for cucurbit seeds. Using this method, nucleic acids extracted from seeds yielded strong signals for each target pathogen in multiplex qPCR. The ability to use a general nucleic acid extraction technique with subsequent PCR to detect bacterial, fungal and viral plant pathogens lends itself to a universal system for cucurbit seed health testing.