应用辣根过氧化物酶化学发光法检测毒素原性大肠杆菌方法的建立

ESTABLISHMENT OF A METHOD FOR DETECTION of escherichia coli by enhanced chemiluminescence with horseradish peroxidase

利用活化酶标复合物（HRP－PBQ－PEI＋－NH3＋）直接标记毒素原性大肠杆菌不耐热肠毒素（ETEC－LT）基因片段（0．8kb）作探针。HRP在戊二醛作用下与单链靶DNA形成DNA和酶的共价复合物，与DNA探针结合的辣根过氧化物酶（HRP）催化相应的发光刺，经增强型化学发光反应（ECL）在普通X光胶片上自显影（CPD）检测ETEC－LT。结果可检测到0．03pg的纯质粒DNA和103的ETEC菌细胞，dotblot杂交证明该酶标基因探针仅与产LT肠毒素的ETEC杂交，其余均未见杂交阳性反应。结果表明酶（HRP）标基因探针化学发光法检测ETEC是一种简便、快速、安全，高度敏感和特异的非放射性标记基因探针检测技术。

A 800-bp polynucleotide probe for heat-labile enterotoxin of Escherichia coli (ETEC-LT+)was directly conjugated with the activated horseradish peroxidase complexes (HRP-PBQ-PEI+-NH3+). The HRP-complexes covalently linked to single-stranded DNA probe forming labelled probe-peroxidase complexes with the action of glutaraldelyde, and redcuced the substrate hydrogen peroxide (detection reagent 1), to detect the enterotoxigenic Escherichia coli (ETEC) by Chemiluminescent Photographic Detection (CPD) through Enhanced Chemiluminescence (ECL) recorded by exposure to X-ray film. Slot blot (Dot blot) hybridization demonstrated that the HRP-conjugated probe was able to detect specimens seeded with 103 ETEC(LT+)cells and 0.03pg plasmid (pMM030), and specifically hybridization with the DNA isolated from ETEC that produced heat-labile enterotoxin (LT+). The results in the present study suggest that the detection of ETEC with the HRP-conjugated gene probe is a rapid, safety,simple and convenient nonradioactivity labelled nucleic acid probe detection technology with high sensitivity and specificity.