罗氏沼虾(Macrobrachium rosenbergii)幼体病原肠杆菌PCR检测技术的建立与应用  

Development and Application of the PCR Detection Method of Pathogenic Enterobacters in the Larvae of the Giant Freshwater Prawn,Macrobrachium rosenbergii

作　　者：陈雪峰[1] 杨国梁[1] 高强[1] 夏正龙[1] 濮剑威[1] 慎佩晶[1] 黄振远[1] 

机构地区：[1]浙江省淡水水产研究所国家罗氏沼虾遗传育种中心浙江省淡水水产遗传育种重点实验室,湖州313001

出　　处：《渔业科学进展》2015年第4期99-104,共6页Progress in Fishery Sciences

基　　金：国家科技支撑计划项目(2012BAD26B04);浙江省重大科技专项(2012C12907);浙江省淡水养殖重点科技创新团队项目(2010R50026-02);湖州市重大科技专项(2011ZD2005)共同资助

摘　　要：根据罗氏沼虾(Macrobrachium rosenbergii)幼体培育期主要细菌性病原阴沟肠杆菌omp A基因序列、产气肠杆菌gry B基因序列设计特异性引物,通过对PCR扩增产物进行测序鉴定与特异性和敏感性试验,建立了两种病原菌的PCR快速检测方法,并对发病样品进行了检测。结果显示,设计的阴沟肠杆菌与产气肠杆菌检测引物能分别扩增出与预计大小一致的385 bp和201 bp的特异性片段,与其余供试菌株无交叉反应。两种检测方法的灵敏度分别为103 CFU/ml和102 CFU/ml。罗氏沼虾幼体样品的检测结果与实际发病情况一致,建立的检测方法也可直接对样品进行PCR检测,而无需细菌分离培养。本研究建立的阴沟肠杆菌与产气肠杆菌PCR检测方法具有较高的特异性与灵敏度,可缩短检测时间,该方法的建立对罗氏沼虾幼体病原的快速诊断、分子流行病学的调查及无特定病原(SPF)群体的建立具有重要意义。The current study was to develop a PCR-based method to detect Enterobacter cloacae and Enterobacter aerogenes in Macrobrachium rosenbergii.Two pairs of primers targeted sequences located within the omp A gene of E.cloacae and gyr B gene of E.aerogenes were used to detect E.cloacae and E.aerogenes.Samples collected from infected larvae were detected with the developed PCR method.The expected DNA fragments of 385 bp and 201 bp were from E.cloacae and E.aerogenes,respectively,and no PCR products were amplified from other bacterium.The sensitivity test showed that the detection limits of PCR were 10^3 CFU/ml for E.cloacae and 10^2 CFU/ml for E.aerogenes.In addition,the detection results of larval samples were consistent with the actual case of the infectious disease.In summary,the PCR diagnostic method was specific and sensitive and is a reliable tool for identification of E.cloacae and E.aerogenes in infected samples with a little time and cost,which would play an important role in quick diagnose,epidemiology investigation and SPF populations construction of the giant freshwater prawn.

关 键 词：罗氏沼虾 阴沟肠杆菌 产气肠杆菌 PCR检测 

分 类 号：S941[农业科学—水产养殖]