5种淡水细菌的多重PCR检测方法的建立及其应用

Establishment and Application of Multiplex PCR Assay for Detection of Five Freshwater Bacterial Species

细菌对水产养殖品种危害大,造成的经济损失高。传统的显微镜镜检方法检测细菌误差大,而利用PCR方法鉴定细菌种类特异性强,准确性高。利用多重PCR探索出一种能准确鉴定水产常见细菌。研究表明,利用PCR分别扩增了嗜水气单胞菌Hly A基因,肺炎克雷伯菌Khe基因,无乳链球菌Sip基因,爱德华氏菌Ser C基因和Eip基因以及迟钝爱德华氏菌Muk F基因和Gad B基因,扩增特异性强。并成功地建立了一种同时用于无乳链球菌和爱德华氏菌等两种细菌以及爱德华氏菌、肺炎克雷伯菌、迟钝爱德华氏菌和嗜水气单胞菌等4种细菌的多重PCR检测方法。为了验证多重PCR方法的准确性,采集患病黄鳝病灶部位,分离培养细菌并提取该细菌基因组DNA,用上述两种多重PCR方法检测出患病黄鳝体内感染的细菌主要是肺炎克雷伯菌和嗜水气单胞菌。建立的多重PCR检测方法对水产常见致病菌的快速诊断和分子流行病学的调查具有重要意义,为鱼类的疾病诊断提供了依据。

Detection of bacteria with traditional method of microscope examination leads to high inaccuracy.On the contrary,the PCR method is specific and accurate to identify bacterial species.A multiplex PCR method for accurate detection of conventional bacteria in aquatic animals was developed and used to amplify Hly A gene in Aeromonas hydrophila,Khe gene in Klebsiella pneumoniae,Sip gene in Streptococcus agalactiae,Ser C gene,Eip gene in Edwardsiella ictaluri,and Muk F gene,and Gad B gene in Edwardsiella tarda.The results showed that the amplification was found to be high specificity.A double PCR method for simultaneous detection of S.agalactiae and E.ictalur and a multiplex PCR method for simultaneous detection of A.hydrophila,K.pneumoniae,E.tarda and E.ictalur were developed.In order to verify the accuracy of the multiplex PCR method,the diseased ricefield eel（Monopterus albus）were collected for isolation and culture of bacteria,as well as for DNA extraction from the cultured bacteria.K.pneumoniae and A.hydrophila were detected by multiplex PCR method.And the established multiplex PCR had important significance for rapid detection of aquatic bacterial conventional pathogenic bacterium and molecular epidemiological research.