3种淡水鱼病原菌多重PCR检测方法的建立与应用  

Establishment and Application of Multiplex PCR Assay for Detection of Three Major Freshwater Pathogens

作　　者：胡宗云 杨培民 闫有利 HU Zongyun;YANG Peimin;YAN Youli(Liaoning Key Laboratory for Prevention and Treatment of Aquatic Animal Diseases,Freshwater Fisheries Research Academy of Liaoning Province,Liaoyang 111000,China)

机构地区：[1]辽宁省淡水水产科学研究院,辽宁省水生动物病害防治重点实验室,辽宁辽阳111000

出　　处：《水产科学》2022年第4期605-613,共9页Fisheries Science

基　　金：财政部与农业农村部现代农业产业技术体系建设专项资金资助项目(CARS-45-34);辽宁省农业科学院市厅级项目(2019DD279141);营口市老边区淡水鱼省级科技特派团项目(2019JH5/10100022);辽宁省淡水水产科学研究院优秀青年科技创新基金资助项目(DS-202001).

摘　　要：根据辽宁地区常见淡水鱼病原菌嗜水气单胞菌、迟钝爱德华氏菌、蜡样芽孢杆菌的相关毒力因子,选择特异性较强的嗜水气单胞菌的溶血素(hlyA)基因、迟钝爱德华氏菌的效应蛋白(eseD)基因、蜡样芽孢杆菌非溶血性肠毒素(nheA)基因为分子靶标,分别设计1对特异性引物,建立可同步检测3种菌的多重PCR检测体系。研究结果显示,在3个毒力基因引物浓度均为0.4μmol/L,模板质量浓度为2.5ng/μL,dNTP和酶添加量分别为2.5μL和0.3μL,退火温度为55℃时,各目的片段均可较好地扩增。敏感性试验结果表明,建立的多重PCR对嗜水气单胞菌、迟钝爱德华氏菌和蜡样芽孢杆菌的最低检测量分别为4.8、3.0ng和2.6ng。对临床分离的菌株样本进行检测,与16SrDNA分子鉴定结果的符合率为100%。建立的多重PCR体系检测方法特异、灵敏、快速,可同步完成淡水鱼源的嗜水气单胞菌、迟钝爱德华氏菌和蜡样芽孢杆菌的检测。A multiplex PCR detection system was developed for three major freshwater pathogens,Aeromonas hydrophila,Edwardsiella tarda and Bacillus cereus,from Liaoning province.hly A,ese D and nhe A were selected for the multiplex PCR.The reaction condition was optimized and the specificity and sensitivity were tested for this method.The results showed that all target fragments were amplified clearly when the concentration of all primer pairs was 0.4μmol/L,and the concentrations of genomic DNA of A.hydrophila,E.tarda and B.cereus were 2.5,2.5 and 2.5 ng/μL,as well as dNTP and polymerase were added 2.5μL and 0.3μL,respectively,with the annealing temperature of 55℃.The sensitivity test showed that the multiplex PCR had a high sensitivity with the detection limit of 4.8 ng for A.hydrophila,3.0 ng for E.tarda and 2.6 ng for B.cereus.The coincidence rate of the multiplex PCR method and 16S r DNA identification method for suspicious clinical samples was 100%.The established multiplex PCR method was specific,sensitive and rapid to simultaneously detect the three pathogens from freshwater fishes.

关 键 词：嗜水气单胞菌 迟钝爱德华氏菌 蜡样芽孢杆菌 多重PCR 

分 类 号：S917.1[农业科学—水产科学]