联合应用表型分析、热休克蛋白65限制性片段长度多态性分析和测序鉴定养鱼水中的非结核分枝杆菌

目的了解养鱼水中非结核分枝杆菌(NTM)的分布情况。方法采集30份市售养鱼水标本,通过分离培养和生化反应初步鉴定,然后从培养菌落中提取DNA,PCR扩增65kD分枝杆菌抗原,扩增产物:(1)分别应用两种限制性内切酶BstEⅡ和HaeⅢ酶切,然后进行琼脂糖凝胶电泳和限制性片段长度多态性分析(PRA);(2)直接测序。结果30份市售养鱼水中29份样本分枝杆菌培养阳性,其中6份样本分别生长出两种形态性状完全不同的菌落,共分离出35株分枝杆菌,表型特征均符合NTM。理化性质、PRA和测序鉴定发现,戈登分枝杆菌8株(23%)、龟-偶然分枝杆菌复合群8株(23%)、日内瓦分枝杆菌9株(26%)、不产色分枝杆菌1株,其余9株未鉴定到种。结论NTM广泛存在于市售养鱼水中,分子生物学方法与常规细菌学鉴定可互相补充,提高鉴定的正确性。

Identification of Nontuberculous Mycobacteria in Fish Tank Water by Phenotyping and PCR-RFLP and DNA sequencing of Mycobacterial hsp65 gene

Objective To investigate NTM distribution in fish tank water in Beijing area. Methods A total of 30 fish tank water samples were collected and incubated for NTM growth in Lowenstein-Jensen media at 28℃ and 37℃,respectively.NTM were further identified by three methods:(1)traditional phenotypic methods-growth and biochemical characteristics;(2)restriction fragment length polymorphism(RFLP) of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein(hsp65);(3) PCR-sequencing. Results Thirty-five NTM strains were isolated in 29 out of 30 fish tank water samples.Traditional phenotypic Methods and genotypic characterization(RFLP and sequencing) revealed that 8 strains(23%) were M.Gordonae,8 strains(23%) belonged to M.fortuitum complex(2 strains M.fortuitum and 2 strains of M.peregrinum),9 strains(26%) were M.genavense, 1 strain was M.nonchromogenicum,and 9 strains were unidentified NTM. Conclusion NTM were wildly distributed in fish tank water that might be one of major sources for NTM infection.PCR-based molecular Methods showed high sensitivity and specificity in identification of NTMs.