16S rRNA在常见致病分枝杆菌菌种鉴定中的应用

Application of 16S rRNA in identification of common pathogenic Mycobacteria

目的通过分析16S rRNA在不同分枝杆菌中的异同,探讨测序技术有关的实验和数据处理问题。方法采用Mega6.0软件对34株非结核分枝杆菌分离株及美国国家生物技术信息中心(NCBI)现有的55株常见致病分枝杆菌和1株诺卡菌的16S rRNA序列进行比对分析,并用Maximum Likelihood法构建进化树,最后用DNASTAR/MegAlign程序计算种间及种内相似性。结果PCR扩增后获得长度约为1500 bp的目的片段,与下载获得的16S rRNA序列进行比对分析并剪齐后保留1248 bp,16S rRNA可以将常见致病性分枝杆菌分为18群,种间相似性为91.5%~100.0%,种内相似性为99.0%~100.0%。结论16S rRNA种内保守性高且具有较好的种特异性,除了龟分枝杆菌和脓肿分枝杆菌、鸟分枝杆菌和胞内分枝杆菌、堪萨斯分枝杆菌和胃分枝杆菌、溃疡分枝杆菌和海鱼分枝杆菌、人型分枝杆菌和牛型分枝杆菌不能分开外,16S rRNA可将常见致病性分枝杆菌各种分开,可作为一种较好的鉴定分枝杆菌的方法。

Objective To explore the experimental and data processing problems related to sequencing technology by analyzing the similarities and differences of 16S rRNA in different Mycobacteria.Methods The Mega6.0 software was used to conduct the comparative analysis on 16S rRNA sequence in 34 isolated strains of non-tuberculosis Mycobacterium,55 existing strains of common pathogenic Mycobacteria and 1 strain of Nocardia farcinica in NCBI.The Maximum Likelihood method was used to construct the evolutionary tree,and the DNASTAR/MegAlign program was finally used to calculate the interspecific and intraspecific similarity.Results The 1500 bp target fragment was obtained after PCR amplification,after cutting to the same length,1248 bp was retained,16S rRNA could divide the common pathogenic Mycobacteria into 18 groups,the range of interspecific similarity was 91.5% to 100.0%,and the range of intraspecific similarity was 99.0% to 100.0%.Conclusion 16S rRNA has high intraspecific conservatism and good intraspecific specificity,which can well distinguish various kinds of the common pathogenic Mycobacteria,except for Mycobacteria chelonae and Mycobacteria abscessus,Mycobacteria avium and Mycobacteria intracellular,Mycobacteria kansasii and Mycobacteria gastri,Mycobacteria ulcerans and Mycobacteria marinum,Mycobacteria bovis and Mycobacteria tuberculosis.