Abstract: Objective To examine DNA sequence polymorphisms in the 16S~23S rDNA gene internal transcribed spacer(ITS) regions of five emerging species: Nocardia beijingensis, N. blacklockiae, N. thailandica, N. elegans and N. vinacea and to screen specialized Nocardia ITS probes for epidemiological study. Methods A set of six isolates belonging to the species of interest was studied. A PCR-based reverse line blot(RLB) hybridization assay incorporating species, or intraspecies ITS rDNA gene operon-specific probes was then developed for species identification. Results Substantial intraspecies sequence variation among different ITS operons was identified. Four sequence types of N. thailandica, eight types of N. beijingensis, and five sequence types of N. blacklockiae, N. elegans and N. vinacea were found. A number of unique RLB pattern types were observed for the cloned products of 2 N. beijingensis species including 4 RLB types, N. blacklockiae 3, N. elegans 3, N thailandica 4 and N. vinacea 2. Conclusion The results represent the first evidence of ITS sequence heterogeneity in emerging species of Nocardia. The PCR/RLB assay demonstrated high specificity and showed promise in both the identification and genotyping of Nocardia species. All N. beijingensis-ITS, N. blacklockiae-ITS and N. elegans-ITS probes were highly specific.

Key words: Nocardia, 16S~23S rDNA gene internal transcribed spacer regions, internal transcribed spacer sequence type, reverse line blot hybridization, internal transcribed spacer reverse line blot type