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

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

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。

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.