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目的对潍坊地区喷泉水中检测出的1株军团菌进行分子生物学鉴定和系统发育分析,为潍坊市军团菌病的预防和控制提供有益信息。方法于2016年7~9月采集潍坊市不同水源的水样,采用抽滤浓缩法浓缩获得细菌浓缩液,用细菌基因组试剂盒提取浓缩菌液中的总细菌基因组DNA,使用针对军团菌属16SrRNA基因片段的特异性引物PCR特异性扩增军团菌属16SrRNA基因片段并测序,与从GenBank获得的另外31株军团菌的16SrRNA的基因序列进行对比分析,采用MrBayesv3.2软件对16SrRNA基因片段序列数据矩阵进行贝叶斯分析并绘制基因进化树。结果 PCR扩增潍坊市水样军团菌属特异性16SrRNA基因片段(386bp),其中一喷泉水样阳性。测序分析该菌株(命名为WF-S)的16SrRNA基因序列与最新发现的沙特军团菌16SrRNA基因序列的遗传距离较近,相似度达99.4%,在构建的贝叶斯基因进化树中与沙特军团菌以其后验概率0.89聚类为一枝。结论 WF-S为一株沙特军团菌,在潍坊地区首次发现,也是国内首次报道的沙特军团菌。
Objective To identify and phylogenetically analyze a strain of Legionella detected in fountain water in Weifang, providing useful information for the prevention and control of Legionella in Weifang. Methods The water samples from different water sources in Weifang City were collected from July to September in 2016. The bacterial concentrate was concentrated by suction filtration and concentration method. Total bacterial genomic DNA was extracted from the bacterial suspension by bacterial genomic kit. Gene fragment specific primers PCR specific amplification of Legionella 16SrRNA gene fragment and sequencing, and GenBank access to the other 31 strains of Legionella 16SrRNA gene sequence comparison analysis using MrBayesv3.2 software 16SrRNA gene fragment sequence data Matrix Bayesian analysis and mapping of phylogenetic tree. Results The 16S rRNA gene-specific 16S rRNA gene fragment (386 bp) was amplified by PCR from Weifang City. Among them, a fountain water sample was positive. Sequencing analysis The 16S rRNA gene sequence of this strain (named as WF-S) has a close genetic distance to the newly discovered 16S rRNA gene sequence of Legionella pneumophila, with a similarity of 99.4%. In the constructed Bayesian phylogenetic tree, The bacteria clustered as a branch with its posterior probability 0.89. Conclusion WF-S is a Legionella pneumophila, first discovered in Weifang region and also the first case of Legionella pneumophila in China.