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

Abstract

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 165 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-specif ic by specificity testing. N. albus NSPOIT, N. jensenii NSPlgT, N. plantarum NSP21T and N. simplex NSP2ZT 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 NSPOIT. The additional 17 strains of N . albus and the additional four strains of N. simplex gave PCR products identical to those of N. albus NSPOIT and N. simplex NSP2ZT, respectively. Multiplex PCR was found to be rapid, species-specif ic and reproducible. The technique evaluated in this study proved to be effective for rapidly identifying Nocardioides strains to species level.

Marmoricola aurantiacus gen. nov., sp. nov., a coccoid member of the family Nocardioidaceae isolated from a marble statue.

“…Despite its phylogenetic proximity to the genus Aeromicrobium, the fatty acid profile and all other chemotaxonomic features characterize N. jensenii as a true member of the genus Nocardioides (Schumann et al, 1997 ;Suzuki & Komagata, 1983 ;. Strain BC 361 T differs from its closest phylogenetic neighbour N. jensenii not only by chemotaxonomic characteristics but also by sequences of the PCR primer designed for rapid identification of N. jensenii by multiplex PCR (Park et al, 1998) and of 16S rDNA of strain BC 361 T . The nucleotide pair G-G at position 445 and 446 in N. jensenii is replaced by the pair C-A in the sequence of strain BC 361 T .…”

“…The predominance of iso-C "':! is characteristic for the genus (Yoon et al, 1997 ;Park et al, 1998). The polar lipid profiles include phosphatidylglycerol, diphosphatidylglycerol and two α-glycol positive phospholipids (O'Donnell et al, 1982).…”

Multiplex PCR assay for rapid identification of three endangered snake species of India

“…DNA sequencing is a useful approach (Parson et al 2000;Jerome et al 2003) but proves to be costly and time-consuming one (Purcell et al 2004). On the other hand, PCR techniques coupled with novel primers designed from target genes have enabled rapid species-specific identification, and help overcome the problems of DNA sequencing (Park et al 1998;Guha and Kashyap 2005). Such tests could be multiplexed for two or B. Dubey Á P. R. Meganathan Á I. Haque (&) National DNA Analysis Centre, Central Forensic Science Laboratory, 30-Gorachand Road, Kolkata, West Bengal 700 014, India e-mail: haque_cfslk@yahoo.co.in more PCR reactions simultaneously in the same tube so as to identify several species in a single step, rapidly.…”