应用复合引物扩增大肠杆菌肠毒素基因的研究

Amplification and detection of enterotoxigenic Esherichia coli genes Using polymerase chain reaction

以两对合成的不同寡核苷酸引物通过聚合酶链反应（ＰＣＲ）、扩增肠毒素大肠杆菌（ＥＴＥＣ）不耐热肠毒素（ＬＴ）和耐热肠毒素（ＳＴ）基因；两对引物从ＥＴＥＣＬＴ和ＳＴ基因中分别扩增出３１４ｂｐ，２３７ｂｐ的ＤＮＡ片段，均能与相应的ＬＴ和ＳＴ基因探针杂交。ＬＴ扩增产物（３１４ｂｐ）和ＳＴ扩增产物（２３７ｂｐ）分别和ＳｍａⅠ和ＨｉｎｃⅡ酶切后，产生１９０ｂｐ和１２４ｂｐ，１４７ｂｐ和９０ｂｐ的ＤＮＡ片段。同一扩增反应中应用ＬＴ和ＳＴ复合引物进行扩增，３种基因型ＬＴ、ＳＴ和ＬＴＳＴＥＴＥＣ从样品中鉴定出。１０９份动物腹泻粪样分别用ＰＣＲ，核酸杂交和ＥＬＩＳＡ进行测定，结果表明，ＰＣＲ是最为灵敏和快速的测定ＥＴＥＣ的方法。

Two different sets of oligonucleotide primers synthesized were used to amplify the enterotoxin genes of heat-labile (LT) and heat-stable(ST) enterotoxins of ETEC.314 and 237bp DNA fragments were amplified by LT and ST primers from LT and ST gene,respectively.The 314 bp(LT) and 237bp (ST) DNA fragment were digested to 190 and 124bp.147 and 90bp DNA fragments by SmaⅠ and HincⅡ.respectively.Three types of ETEC strains corresponding to LT、ST and LTST genetype were distinguished by the same procedure of PCR using the mixture of two sets of primers.109 stool specimens from diarrheal animals were examined by PCR、hybridization and ELISA.The results showed that PCR was the most sensitive method among them.