Progress Towards a Real‐time PCR Assay for the Simultaneous Detection of Clavibacter michiganensis subsp. michiganensis and Pepino mosaic virus in Tomato Seed

Abstract Quarantined phytopathogens such as Pepino mosaic virus (PepMV) and Clavibacter michiganensis subsp. michiganensis can be introduced into tomato transplant houses and fields on infested seed and thereby cause significant economic losses. Hence, specific and sensitive seed health assays are required to exclude these organisms. Currently, separate assays must be conducted on seed samples for each pathogen, making the process time‐consuming and expensive. One approach to improve the efficiency of seed health testing is multiplex real‐time PCR; however, PCR can be inhibited by compounds co‐extracted from seed tissues with nucleic acids. To address this concern, we explored the use of magnetic capture hybridization (MCH) to concentrate and purify target nucleic acids prior to real‐time PCR. The combination of MCH with multiplex real‐time PCR resulted in a 10 2 –10 3 ‐fold increase in detection sensitivity for both pathogens compared to DNA extraction and direct multiplex real‐time PCR. The detection threshold observed for the MCH multiplex real‐time PCR assay was a combination of 10 5 C. michiganensis subsp. michiganensis CFU/ml plus a 10 −4 ‐fold dilution of total RNA extracted from PepMV‐infected tomato leaf tissue. These observations provide proof for the concept that MCH can facilitate the simultaneous detection of Clavibacter michiganensis subsp. michiganensis and PepMV by multiplex real‐time PCR.