PCR技术制备ETEC耐热性肠毒素Ⅰ基因探针及其初步应用

Preparation and Preliminary Utilization ofGene Probe of Heat stable Enterotoxin I(STI) of Enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR)

产肠毒素性大肠杆菌(ETEC)耐热性肠毒素I(STI)重组质粒pSLM004酶切TaqI片段作为模板,经(PCR)技术扩增STI基因,标以α-^(32)p-dATP作STI基因探针。菌落原位杂交结果表明该探针特异性高、敏感性强。通过对180株婴幼儿腹泻病料和52株仔猪腹泻物分离的大肠杆菌(E.coli),以及28株已知血清型的E.Coli标准菌株菌落原位杂交检测,结果分别有11、2和2株为杂交阳性反应,阳性率分别为6％(11/180),4％(2/52),和7％(2/28);而对84株从犊牛腹泻物分离的E.coli中,未检出杂交阳性株。乳鼠生物学试验比较研究表明,15株探针检测阳性菌株中13株乳鼠试验也为阳性反应,两者符合率为87％(13/15)。

The Heat stable enferotoxin I(ST) recombination plasmid pSLM004 of ETEC was cleaved with restriction endonuclease Taq I,the fragment of Taq I was labled with α-32p-DATP by PCR to be utilized as STI gene probe.Hybridization of colony in situs on filters indicated that the probe wsa highly specific and sensitive.Amount of 180 strains of E.coli which isolated from children's diarrhea and 52 strains from piglets,28 standard strains of E.coli serum types were identified,11,2 and 2 were positive,respectively,the hybridization positive rate was 6%,4% and 7% respectively,but 84 strains E.coli which isolated from calf with diarrhea disease,none was positive when detected with the STI probe.Among 15 STI gene probe hybridization positive E.coli strains,13 of them showed positive in suckling mouse bioassay,the coincidence ratio of the two assay was 87%.