Application of multiplex PCR using species-specific primers within the 16S rRNA gene for rapid identification of Nocardioides strains

For the rapid identification of Nocardioides strains, multiplex PCR, using 16S rDNA as target gene, was used and its value was evaluated. Forward primers specific for Nocardioides albus, Nocardioides jensenii, Nocardioides plantarum and Nocardioides simplex, among the five validly described Nocardioides species, were designed from the alignment of 16S rDNA sequences. Nocardioides luteus has been shown to be a member of the same species as N. albus by recent molecular systematic studies and preliminary DNA-DNA relatedness tests. Therefore, N. albus and N. luteus were considered as members of the same species in this study. Each primer was found to be species-specific by specificity testing. N. albus NSP01(T), N. jensenii NSP19(T), N. plantarum NSP21(T) and N. simplex NSP22(T) could be clearly differentiated by PCR products characteristic for each species in the multiplex PCR assay. N. luteus gave an identical result to N. albus NSP01(T). The additional 17 strains of N. albus and the additional four strains of N. simplex gave PCR products identical to those of N. albus NSP01(T) and N. simplex NSP22(T), respectively. Multiplex PCR was found to be rapid, species- specific and reproducible. The technique evaluated in this study proved to be effective for rapidly identifying Nocardioides strains to species level.