摘要
为建立一种能同时鉴别鸭瘟病毒(DPV)和番鸭细小病毒(MDPV)的双重PCR方法,根据DPV UL6基因和MDPV NS-VP1基因分别设计并合成两对特异性引物,通过对双重PCR反应的引物浓度和退火温度优化,建立了能快速检测DPV和MDPV的双重PCR方法,并对该方法的特异性、敏感性和重复性进行验证。结果表明:设计的两对特异性引物均能扩增出目的条带,其大小分别约为376 bp和624 bp;优化引物浓度组合为:DPV引物1.0μmol/L,MDPV引物2.5μmol/L,最佳退火温度为52.4℃。DPV和MDPV的核酸最低检出量分别为4 pg和173 pg,建立的双重PCR分别对鹅细小病毒、禽流感病毒、新城疫病毒、鸭疫里默氏杆菌、沙门菌、大肠杆菌、葡萄球菌、鸭源巴氏杆菌核酸扩增结果均呈阴性;对实验室保存的9份临床病料检测结果DPV检出率33.3%(3/9),MDPV检出率55.6%(5/9)。综上所述,建立的双重PCR方法特异性强、敏感性和稳定性较好,为DPV和MDPV的临床病料检测提供了技术支撑。
In order to establish a double polymerase chain reaction(PCR)for detection of duck plague virus(DPV)and Muscovy duck parvovirus(MDPV),according to DPV UL6 gene and MDPV NS-VP1 gene,two pairs of specific primers were designed and synthesized respectively.By means of optimization of double PCR primer concentrations and annealing temperature and specificity,sensitivity and repeatability of double PCR were established and verified.The results showed that the designed two pairs of specific primers could amplify the target bands,with the sizes of 376 bp and 624 bp respectively.Optimal concentration of primers combination were 1.0μmol/L DPV primer,2.5μmol/L MDPV primer,52.4℃annealing temperature.The minimum nucleic acid detection amounts of DPV and MDPV were 4 pg and 173 pg,respectively,and the double PCR results were negative for the nucleic acid amplification of goose parvovirus,avian influenza virus,Newcastle disease virus,Riemerella anatidis,Salmonella,Escherichia coli,Staphylococcus,pasteuris-derived duck bacilli,respectively.The detection rates of DPV and MDPV were 33.3%(3/9)and 55.6%(5/9),respectively.In summary,the established double PCR method had good specificity,sensitivity and stability,providing technical support for the detection of clinical disease materials of DPV and MDPV.
作者
吴专伟
饶体宇
鲜思美
彭庆波
张友
包细明
李婷
张益
吴伯梅
WU Zhuanwei;RAO Tiyu;XIAN Simei;PENG Qingbo;ZHANG You;BAO Ximing;LI Ting;ZHANG Yi;WU Bomei(College of Animal Science,Guizhou University,Guiyang,Guizhou 550025;Institute of Animal Disease Research of Guizhou,Guiyang,Guizhou 550025)
出处
《中国家禽》
北大核心
2019年第12期17-21,共5页
China Poultry
基金
贵州大学大学生创新创业训练计划项目[贵大国创字(2018)014号]
贵州省农业攻关项目[黔科合支撑(2016)2506号]
作者简介
吴专伟(1995-),男,本科生,研究方向为预防兽医,E-mail:2437461602@qq.com;通讯作者:鲜思美(1974-),女,副教授,博士,主要从事预防兽医学的教学与研究工作,E-mail:xiansimei2005@163.com
