PCR法制备地高辛标记DNA探针斑点杂交检测副溶血性弧菌tlh基因

Detection of the tlh Gene of Vibrio Parahaemolyticus by Dot-blot Hybridization

目的建立检测副溶血性弧菌种特异性tlh基因的斑点杂交方法。方法采用PCR法制备地高辛标记DNA探针,建立斑点杂交条件。杂交反应条件优化如下:杂交温度65℃,杂交时间9～12h,探针使用浓度100～125pg/ml。结果地高辛标记tlh基因探针长度为450bp,标记产量为50μg/ml。探针具有较高的灵敏度和特异性,可重复使用4次。结论本方法具有快速、简便、高效的特点,适用于副溶血性弧菌菌株的鉴定。

Objective To establish a dot-blot hybridization method to detect the species-specific tlh gene of Vibrioparahaemolyticus. Method PCR was carried out to prepare the digoxingenin （DIG） -labeled DNA probe, and the dot-blot technique was established subsequently. The dot-blot reaction conditions were optimized as follows： the hybridization was allowed at 65 ℃ for 9 to 12 h, and the concentration of probe in the final hybridization reaction were from 100 to 125 pg/ml. Results The DIG-labeled DNA fragment was 450 bp in length at a concentration of 50 μg/ml. The DNA probe gave high sensitivity, specificity and repeatability. Conehtsion This method was rapid, simple and effective for the detection of the isolated strains of V. parahaemolyticus.