摘要
根据GenBank中的序列,合成针对鸭圆环病毒(DuCV)REP基因、鹅细小病毒(GPV)NS1基因、鸭肠炎病毒(DEV)UL6基因的3对特异性引物,以病毒的克隆质粒作为模板,进行退火温度、引物终浓度的优化,建立一种能同时鉴别DuCV、GPV、DEV的三重PCR检测方法,并检验该方法的特异性、敏感性和重复性。结果显示:试验所建立的三重PCR检测方法的最适退火温度为58.5℃,引物DuCV REP、GPV NS1、DEV UL6的最适终浓度分别为0.9、0.6、0.7μmol/L;该方法能够同时扩增出DuCV、GPV和DEV的特异性片段,而不能扩增出NDV、RA、AIV、DHV和TUMV,表明该方法特异性强;该方法对质粒DuCV、GPV和DEV模板的最低检测量分别为1、100、10 fg,表明该方法敏感性好;运用该方法对DuCV+GPV+DEV、DuCV+GPV、GPV+DEV、DuCV+DEV、DuCV、GPV、DEV进行检测,均能扩增出与预期一致的特异性片段,表明该方法重复性好;运用该方法对42份临床样本进行检测,其检出率分别为26.19%、30.95%和19.05%,与单一PCR的检出结果一致,表明该方法能适用于临床样本的检测。试验建立的方法具有快速、简便、特异性强、敏感性好、重复性好等特点,可用于DuCV、GPV和DEV临床样本混合感染的快速诊断及鉴别诊断。
According to the sequence in GenBank,3 pairs of specific primers were synthesized for duck circovirus(DuCV)REP gene,goose parvovirus(GPV)NS1 gene,duck enteritis virus(DEV)UL6 gene,and the virus clone plasmid was used as a template.The annealing temperature and final primer concentration were optimized to establish a triple PCR detection method that can simultaneously identify DuCV,GPV and DEV,and test the specificity,sensitivity and repeatability of the method.The results showed that the optimal conditions included the annealing temperature 58.5℃and the optimal primer concentrations for DuCV REP,GPV NS1,DEV UL60.9,0.6,0.7μmol/L,respectively.This detection method were sensitive to DuCV,GPV and DEV,but cannot amplify NDV,RA,AIV,DHV and TUMV,indicating that the method has strong specificity;the minimum detection volumes of this method for plasmid DuCV,GPV and DEV templates were 1,100,10 fg,indicating that the method is sensitive;using this method to detect DuCV+GPV+DEV,DuCV+GPV,GPV+DEV,DuCV+DEV,DuCV,GPV,DEV,amplification of specific fragments consistent with expectations indicates that the method has good reproducibility.42 clinical samples were detected by this method,and the detection rates were 26.19%,30.95%,and 19.05%,which were comparable to those of a single PCR,indicating that the method can be applied to the detection of clinical samples.The method established in this study has the characteristics of rapidness,simplicity,strong specificity,good sensitivity and good reproducibility.It can be used for the rapid diagnosis and differential diagnosis of mixed infection of DuCV,GPV and DEV clinical samples.
作者
田琴
张明华
周碧君
程振涛
王开功
文明
TIAN Qin;ZHANG Minghua;ZHOU Bijun;CHENG Zhentao;WANG Kaigong;WEN Ming(College of Animal Science,Guizhou University,Guiyang,Guizhou 550025,China;Guizhou Animal Disease Research Laboratory,Guiyang,Guizhou 550025,China;Guizhou Province Animal Biological Products Engineering Technology Research Center,Guiyang,Guizhou 550025,China)
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2021年第2期225-230,共6页
Journal of Hunan Agricultural University(Natural Sciences)
基金
国家自然科学基金项目(31560703)
贵州省百层次创新型人才项目(黔科合人才[2016]4009号)
贵州省科技平台及人才团队计划项目(黔科合平台人才[2018]5253号)
贵州省生态家禽产业技术体系功能实验室项目(黔农体系[2017]03号)
三穗鸭工程技术研究中心建设项目(黔科合平台人才[2019]5203号)
贵州省研究生教育创新计划项目(GZZ2017002)。
