多位点串联重复序列分析应用于中国肠炎沙门菌分型能力的评价

Evaluation on application of multi locus variable number tandem repeat analysis in molecular typing of Salmonella enteritidis strains isolated in China

目的比较脉冲场凝胶电泳(PFGE)和多位点串联重复序列分析(MLVA)分型方法用于我国肠炎沙门菌分子分型的能力,建立我国肠炎沙门菌MLVA分型标准操作方法及数据库。方法根据国际PulseNet公布的肠炎沙门菌PFGE和MLVA分型方案,对来自我国6个省(直辖市)的289株肠炎沙门菌进行分子分型分析,并结合流行病学资料,评价这两种分型方法对我国肠炎沙门菌分离株的分型能力。结果 289株肠炎沙门菌经XbaⅠ酶切,PFGE后获得55种带型,其分辨能力(D值)为0.8433。PFGE优势带型为JEGX01.CN0003及JEGX01.CN0001,带型频率分别为35.6%、25.6%,但二者仅表现两个条带的差异,其余型别均低于5%。采用MLVA分析,获得63种型别,分为2个群,D值为0.8608,说明MLVA分辨能力高于单酶切PFGE,但分型能力仍然有限。MLVA主要型别ST1包含了97株菌株,占37.5%,分布于各省及各年份。若联合PFGE及MLVA分型,则产生104种型别,D值为0.9058。对流行病学调查显示为肠炎沙门菌暴发病例菌株进行PFGE双酶切及MLVA分型,结果均显示这些菌株具有明显的聚集性,但不能与同期散发菌株完全区分开。结论 MLVA与PFGE分型方法的分辨能力在肠炎沙门菌中较低,在确认肠炎沙门菌引起的暴发事件时,需紧密结合流行病学调查资料,采用双酶切PFGE或MLVA进行分型分析。

Objective To analyze the discriminatory ability of pulsed - field gel electrophoresis （PFGE） and multi locus variable number tandem repeat analysis （MLVA） in molecular typing of Salmonella enteritidis isolates in China and establish a standard MLVA typing protocol and a subtyping database of S. enteritidis. Methods With the application of standard PFGE and MLVA protocols from PulseNet International network, the molecular typing of 289 isolates of S. enteritidis from 6 provinces in China was condcuted. The respective discriminatory power of the two assays were evaluated combined with the epidemiological information of the strains. Results The chromosomal DNA prepared from each S. enteritidis strain was digested with Xba I restricted endonucleotidase and 55 PFGE patterns were observed. The discrimination index （D） was 0.8433 for PFGE typing. The predominate patterns of PFGE were JEGX01. CN0003 （35. 6% ） and JEGX01. CN0001 （25. 6% ） respectively, and the major MLVA type ST1 was detected in 97 S. enteritidis isolates （37.5%） distributed in all provinces in different years, indicating that the discriminatory ability of MLVA was higher than PFGE. When PFGE and MLVA were integrated, 104 variant patterns were obtained and the D value reached 0. 9058. For some strains from outbreaks, several clusters were detected through PFGE and MLVA analysis, but these strains could be distinguished completely from the sporadic strains detected during the same period. Conclusion The discriminatory powers of MLVA and PFGE are low for S. enteritidis. It is necessary to conduct PFGE and MLVA analysis in epidemiological survey to confirm the outbreak caused by S. enteritidis.