PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band

Abstract

A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp. carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0.55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum, betavasculorum or odoriferum, or from other Erwinia spp. or bacterial genera. The RsaI digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 102 and 1 × 103 colony forming units (CFU) mL-1 and detection sensitivity was increased to as few as 2-4 CFU mL-1 after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.