摘要
目的:建立多重PCR(Multiplex PCR,MPCR)结合变性高效液相色谱(Denaturing High-PerformanceLiquid Chromatography,DHPLC)检测大肠杆菌O157的方法,并了解食品中分离的O157毒力基因携带情况。方法:针对大肠杆菌O157的rfbE、flicH7、eaeA、stx1、stx2基因设计引物,其单一PCR扩增产物在DHPLC上出现的色谱峰为标准色谱峰,建立MPCR-DHPLC检测方法。结果:MPCR扩增产物在DHPLC上的色谱峰,在位置和峰型上与标准色谱峰有较好的对应性。DHPLC能分离大小4个碱基差异的DNA片段。4株分离株检测结果,O157-1出现rfbE、flicH7、eaeA基因色谱峰,O157-2、O157-3、O157-4只出现rfbE基因色谱峰。结论:建立的MPCR-DHPLC方法可用于大肠杆菌O157及其毒力基因的检测。
Objective：A Multi-PCR-DHPLC method for detecting Escherichia coli O157 was developed and the virulence of the E.coli O157 strains isolated from food was studied.Methods： The DHPLC chromatographic profiles of the PCR amplicons of E.coli O157 genes rfbE,flicH7,eaeA,stx1 and stx2 were used as the reference,and the MPCR-DHPLC method was developed.Results： The chromatographic profiles of the MPCR amplicons matched well with the reference.The DNA fragments with size difference as low as 4 bp can be separated by DHPLC.The results of four O157 strains isolated from food showed that the chromatographic profiles of gene rfbE,flicH7 and eaeA appeared in O157-1 strain,whereas only the chromatographic profile of gene rfbE showed up in O157-2,O157-3 and O157-4 strains.Conclusion： The MPCR-DHPLC method established in this study can be used to detect E.coli O157,as well as the virulence genes in these strains.
出处
《中国卫生检验杂志》
北大核心
2012年第5期1057-1059,共3页
Chinese Journal of Health Laboratory Technology
基金
广东省科技计划项目(2009B060600013)
