产肠毒素大肠杆菌双重PCR检测方法的建立

Double PCR for Detection of Virulence Genes K88 and K99 of Enterotoxigenic Escherichia coli Strains

为了快速检测和鉴定产肠毒素大肠杆菌菌毛(K88和K99)基因,本研究设计合成了针对K88、K99的2对特异性引物,对扩增条件进行优化,建立了检测K88和K99的双重PCR方法。该方法对K88、K99基因的扩增产物大小分别为237和314bp;最终确定dNTP终浓度0.4mmol/L,K88、K99的引物终浓度均为25μmol/L,退火温度为52℃。试验结果表明,该方法具有良好的灵敏性和特异性。用所建立的双重PCR方法对实验室分离的23株大肠杆菌进行检测,结果显示,K88单重PCR阳性2株,K99单重PCR阳性3株,K88和K99双重PCR阳性5株。本研究建立的双重PCR检测方法为致幼畜腹泻产肠毒素大肠杆菌的快速准确检测提供了方法。

To detect and identify two fimbriae genes （K88 and K99） of enterotoxigenic Escherichia coli （ETEC）, a double PCR method was developed based on 2 pairs of primers for K88 and K99 genes amplification. 2 different virulence genes of the reference E. coli strain which passed was detected and the double PCR was proved to be specificity and sensitivity. The PCR amplification conditions ultimately were determined that dNTP concentration were 0.4 mmol/L, K88 and K99 primers concen- tration were both 25 μmol/L and the annealing temperature was 52 ℃. Furthermore, a total of 23 samples of E. coli were test- ed by the double PCR assay and the results showed 2 samples were K88, 3 samples were K99, and 5 samples were K88 and K99 positive. The double PCR assay provided a method to detect ETEC which caused diarrhea in piglets.