仔猪腹泻源大肠杆菌的PCR快速检测

Rapid PCR detection of pathogenic Escherichia coli from diarrheic piglets in China

仔猪腹泻是危害养猪业的重要疾病,尽管导致仔猪腹泻的原因非常复杂,但大肠杆菌是其主要病原已经明确。本研究根据GenBank登录的肠毒素ST1、ST2、LT1以及耶尔森氏菌强毒力岛(HPI)的基因序列分别设计特异性引物,建立了检测相应毒力因子的PCR方法。通过对已知毒力型参考菌株的扩增,证明了所建立的PCR方法能够特异鉴定产肠毒素大肠杆菌(ETEC)和HPI+大肠杆菌。采集临床110例仔猪腹泻病例的直肠棉拭子样品,接种LB肉汤于37℃增菌培养4h～6h后,运用所建立的PCR方法进行检测,结果表明有55例(50%)为HPI+大肠杆菌感染,9例(8.18%)为HPI+大肠杆菌与ETEC混合感染,7例(6.37%)为ETEC感染。通过与细菌分离后鉴定的比较,证明该诊断方法具有速度快、检出率高的特点,适合于临床活体检测。

Diarrhea caused by Escherichia coil is the main cause of morbidity and mortality in piglets in commercial farms. The preliminary diagnosis is mainly based on clinical symptom and detailed body dissection. To establish a rapid diagnosis method for porcine diarrhea, specific primers were designed according to the sequences of the enterotoxin ST1, ST2, LT1 and high pathogenicity island （HPI） genes published in the GenBank. Multiplex PCR was performed and specific products of expected size （183 bp for ST1,360 bp for ST2 and 282 bp for LT1, respectively） were detected in the Enterotoxigenic Escherichia coil （ETEC） strains. A PCR product of 280 bp for the HPI gene was detected in the HPI-positive bacterial strains only. A total of 110 feces samples were obtained from live diarrheic piglets from the north region of Jiangsu province and tested by the PCR methods, and fifity-five case （50.0 %） detected HPI-positive; Seven case （6.37 %） were ETEC positive; and nine case （8.18 %） were both HPI- positive E. coil and ETEC positive. The PCR methods used in this study is specific, fast and easy to perform in detection of diarrheagenic E. coli with higher detection rates than the bacterial isolation and identification.