Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis.

Aim Clavibacter michiganensis (Cm) is a seed-borne plant pathogen that significantly reduces tomato production worldwide. Due to repeated outbreaks and rapid spread of the disease, seeds/transplants need to be certified free of the pathogen before planting. To this end, we developed a multiplex TaqMan qPCR assay that can accurately detect Cm in infected samples. Methods and results A specific region of Cm (clvG gene) was selected for primer design using comparative genomics approach. A fully synthetic universal internal control (UIC) was also designed to detect PCR inhibitors and false negative results in qPCR reactions. The Cm primers can be used alone or in a triplex TaqMan qPCR assay with UIC and previously described Clavibacter-specific primers. The assay was specific for Cm and detected upto 10 fg of Cm DNA in sensitivity and spiked assay. Addition of the UIC did not change the specificity or sensitivity of the multiplex TaqMan qPCR assay. Conclusion The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. Significance and impact of the study This assay can be used for biosecurity surveillance, routine diagnostics, estimating bacterial titers in infected material and for epidemiological studies. The UIC is fully synthetic, efficiently amplified, and multiplex compatible with any other qPCR assay.