目的 分析我国分离的大肠埃希菌O157:H7菌株中大质粒pO157的变异情况。 方法 采用限制性内切酶酶切法分析质粒酶切图谱,而后采用嵌套引物进行聚合酶链反应(polymerase chain reaction,PCR)扩增,并用Southern blot进行验证,最后测序。 结果 根据酶切图谱将60株大肠埃希菌O157:H7分为A~E 5个群,而进一步研究表明造成A群与C群HindⅢ 酶切图谱的不同是由C群中的一个小质粒造成的。PCR结果显示A群与C群中的引物对5和25,B群中的引物对25得到的PCR条带与pO157出发菌株不同,其他均相同。测序结果显示造成引物对25在A~C群中与致病性大质粒pO157不同的原因是IS1310序列的插入。 结论 我国大肠埃希菌O157:H7分离株致病性大质粒pO157相对保守,部分菌株中存在插入序列如IS1310,这也是造成我国大肠埃希菌O157:H7 pO157质粒变异的主要原因。
Objective To investigate the evolution of pO157 of O157:H7 strains isolated in China. Methods The classification of the strains was conducted by using restriction enzyme digestion, the amplification by using overlapping primer PCR amplification and the confirmation by using Southern blot. Finally the sequencing was done. Results Sixty strains of E. coli O157:H7 were classified as 5 groups(A-E) by enzyme digestion. However A group and C group were actually belonged to one group because the enzyme digestion difference was caused by a small plasmid in C group using HindⅢ digestion. PCR products from primer pairs 5 and 25 differed between A, C groups and pO157. Only PCR fragments from primer pair 25 showed difference between B group and pO157. The reason for the PCR fragment difference from pair 25 between A-C groups and pO157 was the introduction of IS1310. Conclusion pO157 in the isolated O157:H7 strains in China is relatively conserved. The evolution of pO157 in the O157:H7 strains isolated in China was mainly caused by some inserted sequences such as IS1310.