脉冲场凝胶电泳技术在铜绿假单胞菌现场调查中的应用研究

目的建立铜绿假单胞菌脉冲场凝胶电泳分子分型方法，对某医院患者送检标本及桶装矿泉水中分离到的铜绿假单胞菌进行分子分型，开展分子流行病学分析。方法将铜绿假单胞菌标准株和分离株进行复苏生化鉴定，用SpeI内切酶对试验株菌悬液凝胶块原位消化酶切后进行脉冲场凝胶电泳，比较获得的分子分型指纹图谱，运用Bionumerics5．1聚类分析。结果6株试验菌株中，有3株分离自患者，3株分离自桶装矿泉水，以及1株标准菌种，均获得清晰指纹图谱，条带数在20至25个之间，可分为A、B、C、D、E、F共6个基因型，相似度在70．6％～100．0％之间，其中两株患者来源的菌株相似度为100．O％。结论铜绿假单胞菌基因组经SpeI内切酶酶切后可获得理想的条带数便于分型。患者标本和饮用桶装矿泉水分离株没有直接相关性，但提醒仍需加强桶装矿泉水污染铜绿假单胞菌的管理；脉冲场凝胶电泳技术可有效应用于铜绿假单胞菌遗传学特征、传播途径及分布规律的分子流行病学研究。

Application of pulsed-field gel electrophoresis in field investigation of Pseudomonas aeruginosa

Objective To establish the genotyping method of pulsed-field gel electrophoresis for Pseudomonas aeruginosa, and research into the molecular epidemiological characteristics of clinical isolates from hospital patients and the isolates from mineral water. Methods Standard strains and isolates were recovered and their biochemical identification was carried out.. The suspension of test strains were coated by gel, and total DNA in situ gel block was digested with restriction enzyme Spe I. After running pulsed-field gel electrophoresis, the DNA fingerprint differences were compared to study this stain＇ s relationship by using Bionumerics5.1 cluster analysis. Results The strains including 3 clinical isolates, 3 isolates from mineral water and 1 standard strain occurred clear fingerprint with 20 to 25 electrophoresis bands. Their genotypes were divided into A, B, C, D, E and F with a similarity from 70.6% to 100.0 %, and where-in the similarity of two clinical strains was 100.0%. Conclusion P. aeruginosa genome digesting in situ gel by Spe I endonuclease occurred the ideal number of bands for genotyping. We should strengthen management and pollution survey of P. aeruginosa in mineral water, though the strains from clinical specimens and mineral water were not direct correlation. The genotyping method of pulsed-field gel electrophoresis can be effectively used in molecular epidemiological study including the study of genetic characteristics of P. aeruginosa and investigating it＇ smodes of transmission and state of distribution..