Simultaneous detection of Clavibacter michiganensis subsp. michiganensis and Pepino mosaic virus in tomato seed using magnetic capture hybridization and real time PCR
Johnson, Kameka Latoya
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Pepino mosaic virus (PepMV) and Clavibacter michiganensis subsp. michiganensis are quarantined seedborne pathogens of tomato that cause severe economic losses. Currently, separate seed health assays must be conducted for these pathogens, making the process time consuming and difficult. One method to improve the efficiency of seed health testing is the simultaneous detection of both pathogens using multiplex real time PCR. Magnetic capture hybridization (MCH) was employed to concentrate and purify target nucleic acids from PCR inhibitors present in the seed wash. The combination of MCH with multiplex real time PCR resulted in a 100-1000 fold increase in detection sensitivity compared to direct multiplex real time PCR. The detection threshold of the MCH-multiplex real time PCR assay was 10CFU/ml C. michiganensis subsp. michiganensis with a 10 dilution of total RNA extracted from PepMV-infected tissue. The assay also detected C. michiganensis subsp. michiganensis in 1:10 dilution of naturally infested tomato seed.