变性高效液相色谱检测霍乱弧菌

Identification of Vibrio cholerae by denaturing high-performance liquid chromatography

变性高效液相色谱(DHPLC)是近几年刚发展起来的一种快速检测PCR扩增产物的新技术.应用该技术快速检测O1、O139、非O1、非O139群霍乱弧菌的外膜蛋白基因(ompW).根据霍乱弧菌外膜蛋白基因的特异性引物,PCR扩增的产物经DHPLC进行快速检测.以54株参考菌株做特异性检测.在丹东口岸国境外环境水体采集水样310份,以PCR-DHPLC和常规分离培养进行了比较,对丹东口岸国境外环境水体霍乱弧菌感染状况进行了检测,并对分离株进行了ompW基因测序.试验结果表明该方法有很好的特异性,310份水样中经PCR-DHPLC检测出58株霍乱弧菌的分菌株,阳性率为19%,与常规分离培养比较,符合率为100%.分离株的ompW序列与Genbank中的存在1%～3%的差异.这种检验方法特异性强,简便快捷,为霍乱防治提供了一种有效的检测新手段.

Denaturing high-performance liquid chromatography （DHPLC） is a new technique recently developed for rapid screening of nucleotide polymorphisms in PCR products. We identify the OUter Membrane Protein Gene （ompW） in O1,O139,non-O1 and non-O139 Vibrio cholerae by this technique. Primers specific for the ompW gene in Vibrio cholerae were selected to conduct the DHPLC assays. The specific testing was performed with 54 strains of reference bacteria for the sensitivity testing. 310 water samples were collected from Outside Frontier Water at Dandong Port by PCR-DHPLC technology and the results were compared with that of the routine bacterial separation culture method, and the infection status of O1,O139,non-O1 and non-O139 Vibrio cholerae from Outside Frontier Water at Dandong Port was delected, and ompW gene of separation strains was measured. The results indicated that PCR-DHPLC technology was very specific. 58 strains Vibrio cholerae were detected by PCR-DHPLC, and the total positive rate of Vibrio cholerae in 310 water samples was 19%. The detection results were 100% in accord with that of the routine bacterial separation culture method. There exists 1%-3% difference between ompW gene sequence of separation strains of Vitrrio cholerae and Genbank＇s. It is a quick and useful method for identification of O1,O139,non-O1 and non-O139 Vibrio cholerae.