直接从粪便中检测致犊牛腹泻产肠毒素大肠杆菌的双重PCR方法的建立与应用

Direct detection of the enteritoxigenic Escherichia coli in bovine feces by multiplex PCR

目的产肠毒素大肠杆菌(Enterototxigenic,Escherichia Coli,ETEC)是造成人和动物腹泻的主要病原之一,也是造成新生乳用犊牛腹泻的主要原因,一株ETEC可能产生一种或者多种肠毒素。根据产肠毒素大肠杆菌产生的两种主要毒素的基因序列,设计了两对引物,建立多重PCR方法。该方法仅用4.5小时即可检测出导致犊牛腹泻的产肠毒素大肠杆菌菌株,具有敏感度高、特异性强和检测速度快等特点。本试验的目的是用所建立的多重PCR方法来确定ETEC在导致犊牛腹泻中的作用。采用该方法对乳用犊牛的203个腹泻样品进行了检测,结果,用传统的分离培养方法得到的203株典型大肠杆菌中有135株为ETEC;其中LT阳性为105株,ST阳性为126株,LT+ST阳性的为96株,阳性率为66.5%。而采用直接从粪样中提取PCR模板的方法进行PCR,检测到146个犊牛粪样为ETEC阳性,其中LT阳性为112株,ST阳性为137株,LT+ST阳性为103株,阳性率为71.9%,明显高于传统的检测方法;并且所检测到的146个ETEC阳性的犊牛粪样完全包含用传统的分离培养方法得到的135个阳性粪样,且基因分型结果相同。

Two pairs of primers were designed according to the gene sequences of two main toxins produced by enterotoxigenic Escherichia coli （ETEC） and were used in multiplex PCR to detect ETEC in bovine feces. The result revealed that this method of detecting was a very sensitive, specific and rapid one used for the epidemiological investigations of E. coli isolated from calf with diarrhea. It was proved that this method processed a characteristic features with high sensitivity and specificity as well as the rapid speed of performance. Of the 204 strains of typical E. coli isolated from fecal samples of milk calf, 135 strains was proved to be ETEC, in which 105 strains possessed genes of the heat-liable toxin （LT）, 126 strains possessed genes of the heat-stable toxin （ST）, and 96 strains possessed both types of enterotoxins. However, of these 204 strains of E. coli, 146 strains were positively detected by the multiplex PCR directly from the fecal specimens of milk calf, in which 112, 137 and 103 strains were positive for genes of LT, ST and LT＋ ST respectively.. It is evident from the results of present study that the in-house method to extract the ETEC DNA directly from milk calf for use in multiplex PCR is more sensitive than the conventional culture method commonly used.