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

Abstract

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. 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 qPCRs. The Cm primers can be used alone or in a triplex TaqMan qPCR assay with UIC and previously described Clavibacter primers. The assay was specific for Cm and detected up to 10fg of Cm DNA in sensitivity and spiked assays. Addition of the UIC did not change the specificity or sensitivity of the multiplex TaqMan qPCR assay. The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. This assay can be used for biosecurity surveillance, routine diagnostics, estimating bacterial titres in infected material and for epidemiological studies. The UIC is fully synthetic, efficiently amplified and multiplex compatible with any other qPCR assay.