副溶血性弧菌ERIC-PCR分型及毒力基因检测研究

Genotyping of Vibrio parahaemolyticus by ERIC-PCR and virulence gene detection

目的:建立副溶血性弧菌ERIC-PCR分子分型技术,分析副溶血性弧菌标准菌株及分离株基因组DNAERIC-PCR指纹图谱,并对副溶血性弧菌毒力基因进行检测,以了解不同来源副溶血性弧菌毒力基因携带情况。方法:提取副溶血性弧菌基因组DNA,以肠杆菌基因间共有重复序列(ERIC)为引物进行PCR扩增,PCR产物经琼脂糖凝胶电泳后用凝胶成像分析仪对图谱进行观察分析,并以相似性系数构建聚类图;通过PCR方法对直接耐热溶血素(TDH)和耐热直接相关溶血素(TRH)进行检测。结果:26株副溶血性弧菌均可扩增产生可重复的DNA指纹图谱,ERIC-PCR可将26株菌分为12个型,分辨力指数为0.926;只在临床分离株中检测到TDH基因,而除一株标准菌株外,所有菌株都未检测到TRH基因。结论:研究显示ERIC-PCR可从分子水平对副溶血性弧菌基因组DNA进行快速指纹图谱分析,同时结合毒力基因检测,能够为副溶血性弧菌食物中毒疾病的预防和流行病学调查提供科学依据。

Objective： To establish the ERIC-PCR technique for genotyping Vibrio parahaemolyticus, study the genomic DNA ERIC-PCR fingerprinting of Vibrio parahaemolyticus standard strains and epidemic isolates, investigate the positive rate of virulence genes of Vibrio parahaemolyticus from different sources. Methods= Genomic DNA of Vibrio parahaemolyticus was abstracted and used as the template for PCR. Enterobacteial repetitive intergenic consensus sequences were used as primers to amplify the target sequences in Vibrio parahaemolyticus genomic DNA, amplification products were separated by agarose gel electrophoresis, and electrophoresis maps were analyzed by gel image analysis system,the cluster dendrograms were made according to the coefficient of similarity^thermostable direct hemolysin（TDH） gene and TDFI-related hemolysin （TRH） gene were detected by PCR. Results. Twenty- six Vibrio parahaemolyticus strains were grouped into 12 patterns with discriminative index of 0.926; The tdh gene was positive in all clinical strains but no trh gene was in all strains except one standard strain.Conclusion： ERIC- PCR could be used for analysing fingerprinting of genomic DNA from Vibrio parahaemolyticus quickly, combining with virulence genes detection, it could be used in preventing food poisoning caused by Vibrio parahaemolyticus, and provided scientific basis for Vibrio parahaemolyticus epidemiology investigation.