脉冲场凝胶电泳和多位点串联重复序列分析应用于中国鼠伤寒沙门菌分型能力的评价

摘要

目的 比较脉冲场凝胶电泳(PFGE)和两种国际多位点串联重复序列分析(MLVA)分型方法用于我国鼠伤寒沙门菌分子分型的能力,并初步确定适用于我国鼠伤寒沙门菌MLVA分型中的VNTR位点.方法 根据国际PulseNet公布的沙门菌PFGE分型方案、MLVA分型方案(包含7个VNTR位点,简称MLVA_PN)及欧洲食源性疾病监测网公布的鼠伤寒沙门菌MLVA分型方案(包含5个VNTR位点,简称MLVA-EU),对来自我国5个省(直辖市)的175株鼠伤寒沙门菌进行分子分型分析,并结合流行病学资料,评价这三种分型方法对我国分离的鼠伤寒沙门菌的分型能力.结果 175株鼠伤寒沙门菌经Xba Ⅰ酶切,PFGE后,获得56种带型,其分辨能力(D值)为0.8823.对其中的主优势带型JPXX01.CN0001菌株55株进一步用第二种内切酶BlnⅠ酶切后,获得6种带型.用MLVA_EU分型,获得96种型别,D值为0.9758.应用MLVA_PN分析,获得98种型别,D值为0.9763.两种MLVA分型方法对菌株的分辨能力几乎相同,且分型结果具有较高的一致性.对流行病学调查显示为鼠伤寒沙门菌暴发患者和食物来源的菌株进行PFGE双酶切及MLVA分型,三种分型方法获得一致性结果,均显示这些菌株具有明显的聚集性.结论 两种MLVA分型方法的分辨能力均高于PFGE,在确认鼠伤寒沙门菌引起的暴发事件时,采用需时较短,操作更方便的5个VNTR位点的MLVA分型方法可满足菌株聚集性分析.%Objective To compare the ability of pulse-field gel electrophoresis (PFGE) and two multiple loci VNTR analysis (MLVA) typing methods in molecular typing of Salmonella Typhimurium isolated in China and to obtain the valuable VNTR loci fit for S. Typhimurium molecular typing in China. Methods With the application of standard PFGE and MLVA including seven VNTR loci (MLVA_PN) protocols on PulseNet International network and MLVA protocol containing five VNTR loci (MLVA_EU) on European surveillance network, 175 isolates of S. Typhimurium from five provinces in China were typed. The molecular typing ability of the three methods were evaluated combined with the epidemiological information of the strains. Results The chromosomal DNA prepared from each S.Typhimurium strain was digested with Xba Ⅰ restricted endonucleotidase and 56 PFGE patterns were observed after pulsefield gel electrophoresis. The discrimination index (D) was 0. 8823 for PFGE typing method. The predominant pattern of PFGE, JPXX01. CN0001of 55 S. Typhimurium isolates were further digested with the second enzyme Bln Ⅰ to achieve additional 6 patterns. For MLVA_EU typing, it was showed 96 variant patterns and the D value reached 0. 9758.For MLVA_PN method, 98 variant patterns were obtained and the D value was 0.9763, which indicated the discriminatory ability of the two MLVA typing methods for S. Typhimurium was similar. In addition, the typing results of 175 S. Typhimurium isolates by MLVA_EU were coincident with that by MLVA_PN. When S. Typhimurium strains isolated from patients and food in outbreaks were analyzed by PFGE with double enzymes digestion, MLVA_EU and MLVA_PN respectively, the results were coincident. Conclusion The discriminatory ability of the two MLVA typing methods were higher than that of PFGE. MLVA_EU containing five VNTR loci with the characterization of timesaving and easy manipulation is suitable for cluster analysis when detecting the outbreaks caused by S. Typhimurium in China.