肠炎沙门氏菌脉冲场凝胶电泳分型研究

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis

目的 为研究肠炎沙门氏菌菌株间的关系,特建立了分子流行病学研究方法脉冲场凝胶电泳分型方法,并对从食品中分离的肠炎沙门氏菌进行了分型研究。方法 1991～2 0 0 1年间,从河南、黑龙江等省份采集食品样品,分离出肠炎沙门氏菌,挑取单个菌落增菌,用溶菌酶、蛋白酶K和TE缓冲液依次提取菌体DNA后,用限制性内切酶XbaΙ消化胶块,然后在CHEFMapper仪上进行脉冲场凝胶电泳,电泳条件:0 . 5×TBE缓冲液、14℃、1%琼脂糖、脉冲时间5s～4 5s、2 6h、6V cm。应用λ噬菌体DNA作为脉冲场凝胶分子量标准。结果 从鸡肉、牛肉、熟肉及蔬菜中分离出30株肠炎沙门氏菌,其中18株来自河南省,绝大多数菌株(2 2株)分离于2 0 0 1年。对所得菌株进行脉冲场凝胶电泳后,结果显示30株肠炎沙门氏菌分为A、B两个型,两型图谱之间有8个条带的差异。A型菌株有19株,占全部菌株的6 3. 3% ,B型菌株有11株,占36 .7%。A型之内又可以分出2个亚型,其中A1型占78 .9% ,A2亚型与A1亚型相比的差别在于,A2型在5 0kb处有一条带缺失。在菌株分型与采集时间和采集地点之间没有相关性,但所有从鸡肉中分离到的菌株均为A1型,提示了某种有待进一步研究的流行病学趋势。结论 在研究肠炎沙门氏菌不同菌株之间的分子流行病学关系方面。

Objective To study the relationship between different strains of Salmonella enteritidis, a molecular epidemiologic analysis method-pulsed-field gel electrophoresis was established and Salmonella enteritidis isolated from food samples were tested. Methods Food samples were collected from henan province, heilongjiang province and other provinces in 1991-2001. Strains were isolated and single colony of each salmonella enteritidis strain from an agar plate was picked and enriched,then the total DNA was extracted in turn with lysozyme solution, proteinase K solution and TE buffer. The agarose pulgs were digested with restriction enzyme XbaΙ overnight at 37℃, followed by pulsed-field gel electrophoresis (PFGE) with a CHEF Mapper system in 0.5×TBE buffer with recirculation at 14℃. DNA macrorestriction fragments were resolved on 1% agarose gels, pulse time were ramped from 5s to 45s during a 26h run at 6V/cm. A lambda ladder pulsed-field gel maker was used as a size standard. Results 30 salmonella enteritidis strains were identified in chicken, beef, cooked meat and vegetables. 18 strains were isolated from henna province. More of the stains (22 strains) were isolated in 2001. Two distinct DNA fragment profiles, PFGE types A and B, were observed, representing 63.3% and 36.7% of the isolates respectively. Type A could be subdivided further into two subtypes. Subtype A1 represented the majority (78.9%) of type A strains and differed from subtype A2 in that the latter lacked a band in the 50kb region. There was no correlation between the strains profiles and their isolation time or region. However, all the strains isolated from the chicken were totally type A1,and showed certain epidemiological trend which can be studied in the future. Conclusion The pulsed-field gel electrophoresis is a good method to study the molecular epidemiological relationship between the different Salmonella enteritidis srtains.