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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 analyze the variation of large plasmid pO157 in Escherichia coli O157: H7 isolated in China. Methods Restriction endonuclease digestion method was used to analyze the plasmid digestion map, and then amplified by polymerase chain reaction (PCR) using nested primers. The PCR products were verified by Southern blot and sequenced. Results Sixty Escherichia coli O157: H7 strains were divided into five groups according to the digestion map, and further studies showed that the difference in HindIII digestion pattern between group A and group C was caused by a small plasmid in group C of. The PCR results showed that the PCR bands obtained from primer pairs 5 and 25 in group A and group C and primer pair 25 in group B were different from the original strain of pO157 and others were the same. The sequencing results showed that the reason for the difference between the primer pair 25 and the pathogenic large plasmid pO157 in group A to C was the insertion of the IS1310 sequence. Conclusion The pathogenicity plasmid pO157 of Escherichia coli O157: H7 is relatively conservative in China. Some of the isolates have inserted sequences such as IS1310, which is also the main reason for the variation of Escherichia coli O157: H7 pO157 in China.