简易菌落斑点杂交法快速检测志贺氏菌ipaH基因的实验研究

Detection of ipaH gene of shigella by simple rapid colony blot hybridization

采用ＰＣＲ扩增技术，制备了Ｄｉｇ标记志贺氏菌多拷贝的ｉｐａＨ基因探针。用该探针通过菌落斑点杂交试验，探索快速、特异、敏感、简单检测志贺氏菌的方法。本法检测灵敏度达５－１０ｐｇＤＶＡ／μｌ。含菌量为３×１０６ＣＦＵ／ｍｌ的粪便上清液直接抽滤点膜２５μｌ杂交结果为阳性。与经ＰＣＲ扩增测定的５０株细菌（其中ｉｐｔａＨ基因阳性菌２５株，阴性菌２５株）进行比较，菌落斑点杂文结果与ＰＣＲ扩增结果完全一致。全部实验可在２０ｈ内完成。

A simple,rapid colony hybridization for ipaH gene of Shigella with a digoxigenin-labelled DNAprobe is described.The probe was prepared by PCR mediated Dig-labelling of the ipaH gene. The probewas applied to blots from stool cultures and from purified DNA. The detection limit was 3×106CFU/ml fe-cal supernants and 5-10pg DNA. This method was applied to detection of 50 strains of enteric bacteria.The results were concordant with those by PCR. The experimental procedure could be completed within 20hours.