鮰爱德华氏菌外膜微孔蛋白N基因PCR快速检测方法的建立  

Rapid Detection for Outer Membrane Porin Protein N Gene of Edwardsiella ictaluri by PCR

作　　者：潘延乐 汪开毓[1,2] 杨洁 阳磊[1,2] 吉莉莉[1,2] 耿毅[1,2] 陈德芳[4] 

机构地区：[1]四川农业大学鱼病研究中心,四川雅安625014 [2]四川农业大学动物疫病与人健康四川省重点实验室,四川雅安625014 [3]宜宾市翠屏区水务局,四川宜宾644000 [4]四川农业大学水产养殖系,四川雅安625014

出　　处：《四川动物》2015年第2期264-269,共6页Sichuan Journal of Zoology

基　　金：教育部<长江学者和创新团队发展计划>创新团队项目(IRT0848);四川省科技厅产业链项目(2014NZ0003)

摘　　要：根据Gen Bank中鮰爱德华氏菌Edwardsiella ictaluri外膜微孔蛋白N(porin N)基因序列(Gen Bank No:NC_012779.2)设计了1对引物,预计目的片段大小为381 bp。通过对反应体系和条件的优化,并进行特异性试验、敏感性试验及人工感染组织样品检测,建立了一种快速检测鮰爱德华氏菌的PCR方法。结果表明,在所检测的鮰爱德华氏菌、迟缓爱德华氏菌、嗜水气单胞菌、温和气单胞菌、杀鲑气单胞菌、豚鼠气单胞菌、嗜麦芽寡养单胞菌、鲁氏耶尔森氏菌、海豚链球菌、不动杆菌、产气肠杆菌、大肠杆菌、拟态弧菌、荧光假单胞菌、弗氏柠檬酸杆菌15种细菌中仅鮰爱德华氏菌扩增出特异性条带;敏感性试验结果显示,该方法最小核酸检出量为9.35×10-3ng·μL-1;同时对人工感染的病料肝脏、细菌基因组DNA、细菌菌液及菌落进行扩增,结果显示4种材料均能检测出大小为381 bp的基因片段。本研究所建立的方法特异强、灵敏度高,适用于鮰爱德华氏菌感染病例的高效、快速检测。Specific primers were designed and conducted to amplify a 381 bp DNA frangment from the gene （GenBank, NC._012779.2） encoding a outer membrane porin protein in Edwardsiella ictaluri. The PCR reaction system and reaction conditions were optimized. The specificity and sensitivity of this assay were tested on both normal and infected samples. The resuits from specific tests showed that the E. ictaluri strain GPY was the only strain that could be detected by this method among all the tested strains. The minimal concentration of DNA that can be detected by this method was 9.35 × 10-3 ng·μL-1. Mo- reover, the liver that artificially infected with E. ictaluri, bacterial genomic DNA, bacterial liquid and bacterial colonies were also tested. The 381 bp DNA fragment could be detected from all these materials. In conclusion, the established PCR in this study was specific, sensitive and rapid for the epidemiological surveillance of E. ictaluri infection in channel catfish. Therefore, a rapid method to detect E. ictaluri by PCR was established.

关 键 词：鮰爱德华氏菌 外膜微孔蛋白基因 PCR 

分 类 号：S941.4[农业科学—水产养殖]