摘要
根据GenBank中的鸭疫里氏杆菌(RA)外膜磷酸盐选择性微孔蛋白(Porin)基因序列设计特异性引物,以血清1、2、6、10、11、13、14和17型RA菌株的基因组DNA为模板均扩增出大小为1 212bp的目的片段,序列比对显示该基因在8种血清型菌株间同源性均大于99.6%。针对保守性最高的357bp片段设计特异性引物,建立了PCR检测方法,对鸭致病性大肠杆菌、鼠伤寒沙门菌、鸭巴氏杆菌及鸭肝炎病毒均未扩增出目的条带,表明该方法具有较高的特异性;敏感性试验显示最小检出量为73.96pg的基因组。应用建立的PCR方法对分离自吉林、河北及北京的8个菌株进行扩增,均在357bp处扩增出清晰的目的条带,与细菌分离鉴定的符合率达100%。本研究为RA的鉴定提供了新方法,为RA外膜微孔蛋白的后续研究提供了理论依据。
Specific primers were designed according to outer membrane phosphate selective Porin pro tein gene sequence of Riemerella anatipestifer available in GenBank. Target fragments of 1 212 bp were successfully cloned from genomic DNA of serotypes 1,2,6,10,11,13,14 and 17 R. anatipestifer strains. Multiple sequence alignment analysis showed the homology of the porin gene between the 8 serotype strains was more than 99.6%. Specific primers were designed for 357 bp fragment to establish a PCR diag nostic method. The 357 bp target fragment was not amplified from genome of duck Pasteurella,duck patho- genic Escherichia coli, Salmonella Typhimurium and duck hepatitis virus,which indicated that the method had a high specificity. The lowest amount of DNA of R. anatipestifer detectable by this method was 73.96 pg. Sharp interest bands with a size of 357 bp were amplified from 8 isolates of R. anatipestifer of Jilin, He- bei and Beijing. Coincidence with identification of bacteria was 100 %. Result provides identification methodfor R. anatipestifer and a theoretical basis for subsequent research of Porin.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第7期723-727,共5页
Chinese Veterinary Science
基金
吉林省教育厅"十二五"科学技术研究项目(201372)
吉林省科技发展计划项目(20110221)
国家现代农业产业技术体系建设专项(CARS-43)
国家星火计划项目(2012GA660003)
