Development of a multiplex PCR assay for the detection of key genes associated with subspecies.

The ability to distinguish among the subspecies of isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of . The PCR assay includes the species-specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. ), and primers targeting a 16S rRNA gene region specific for subsp. . The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of subspecies, and an alternative to biochemical test-based differentiation. A possible relationship was noticed between subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. and subsp. isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.