PCR为基础的反向线点杂交及16S～23S rDNA间区测序分析5种少见类型奴卡菌

Reverse line blot hybridization and DNA sequencing studies of the16S～23S rDNA gene internal transcribed spacer regions of fiveemerging pathogenic Nocardia species

目的探讨5种新出现的奴卡菌种16S～23S rDNA内转录间隔区(internal transcribed spacer,ITS)DNA基因序列的多态性,筛查特异的奴卡菌种逆向线点杂交(reverse line blot,RLB)探针。方法对6例奴卡菌标本进行ITS基因扩增、克隆、测序,设计奴卡菌种特异探针及ITS间区rDNA基因特异的探针,进行以PCR为基础的RLB(PCR/RLB)杂交试验,不同的RLB型进行ITS基因测序分析,以鉴别不同的奴卡菌种及种内分型。结果发现2个Nocardia beijingensis菌株ITS间区有8个基因序列型,4个RLB型;N.thailandica有4个基因序列型及RLB型;N.blacklockiae有5个基因序列型,3个RLB型,其中N-bla RLBⅢ型与所有Nbla-ITS探针杂交没有信号;N.elegans有5个基因序列型,3个RLB型;N.vinacea有5个基因序列型,2个RLB型。结论通过PCR/RLB试验,准确的ITS基因测序认识了新出现的菌种,即N.beijingensis,N.blacklockiae,N.elegans,N.thailandica及N.vinacea。同时建立了可以大量筛查奴卡菌种的PCR/RLB方法,以进行快速临床诊断及流行病学研究。

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.