虾源副溶血弧菌致病基因检测与ERIC-PCR分型

Pathogenic gene detection of Vibrio parahaemolyticus isolated from shrimp and genotyping by ERIC-PCR

为了监测上海市养殖对虾中副溶血弧菌致病基因的携带情况及潜在致病株流行情况,采用PCR方法对上海地区不同对虾养殖单位中分离获得的19株副溶血弧菌分离株及1株标准菌株进行6种致病基因的检测筛查,并采用ERIC-PCR方法进行基因分型分析。结果发现:19株副溶血弧菌中未检测出携带tdh、ORF8基因,只有1株携带trh基因,而tox RS/new基因携带率较高,19株中有14株检出阳性,基因携带率为73.7%。对能够引起对虾急性肝胰腺坏死病(AHPND)的副溶血弧菌特异性质粒基因的检测中发现,AP1和AP2基因均有3株检测出阳性,基因携带率均为15.8%。通过ERIC-PCR分型技术将20株副溶血弧菌分成7个不同的类群,其分辨力指数DI值为0.811,表明ERIC-PCR具有较好的基因分型能力,分型结果发现该方法能够有效区分不同时间段获得的菌株,而对不同地理位置获得的菌株区分并不明显。以上结果表明从养殖对虾中分离得到的副溶血弧菌致病基因携带率总体偏低,但存在一定的流行风险,需要防范一些新的致病株的流行,这些可以为对虾养殖单位中副溶血弧菌致病流行株的预防控制提供相关参考。

In order to monitor the carrying status of pathogenic genes and the prevalence of potential pathogenic strains of Vibrio parahaemolyticus from shrimp in Shanghai,the polymerase chain reaction(PCR)and enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction(ERIC-PCR)were applied to detect pathogenic genes and to make genotyping analysis of 1 standard V.parahaemolyticus strain and 19 V.parahaemolyticus strains isolated from shrimp in Shanghai.The results showed no tdh and ORF8 genes were detected in 19 isolated strains and only 1 strain carried trh gene,while the tox RS/new gene was detected in 14 positive strains and the carrying percentage was 73.7%.The AP1 and AP2 genes were detected in specificity V.parahaemolyticus which could cause acute hepatopancreas necrosis disease(AHPND)and the result showed there were 3 positive strains of both genes,and the gene carrying percentage was both 15.8%.The 20 V.parahaemolyticus strains were divided into 7 groups by ERIC-PCR,and the discrimination index(DI)was 0.811,which indicated ERIC-PCR have a good genotyping capability.According to the results of ERIC-PCR,the method could distinguish strains obtained from different periods of time effectively,while the distinction among different geographical strains was not obvious.These results suggested that the carrying percentage of pathogenic genes in V.parahaemolyticus from shrimp was relatively low,while there was a risk of prevalence of new pathogenic strains.These results will provide reference for the prevention and control of V.parahaemolyticus strains in shrimp farms.