摘要
为建立鸽I型腺病毒(PIAdV-I)的快速检测方法,本研究根据PIAdV-I Fiber 2基因的保守区域设计特异性引物,通过优化反应条件,建立了快速检测PIAdV-I的SYBR Green I荧光定量PCR(qPCR)方法。建立的SYBR Green I qPCR方法的标准曲线结果显示,重组质粒标准品pMD19-Fiber在1×10^(3)拷贝/μL^(1)×10^(8)拷贝/μL时和Ct值之间的线性关系良好,相关系数R~2=0.995,扩增效率为90%。利用建立的qPCR方法对鸽II型腺病毒(PIAdV-II)Fiber 2基因保守区质粒标准品、鸽圆环病毒(PICV)、新城疫病毒(NDV)、多杀性巴氏杆菌(PM)和禽致病性大肠杆菌(APEC)的检测结果均为阴性,仅PIAdV-I检测为阳性,特异性强;该方法对浓度为1×10^(0)拷贝/μL^(1)×10^(8)拷贝/μL的重组质粒标准品进行检测,结果显示最低检测限为1×10^(2)拷贝/μL,而普通PCR最低检测限为1×10^(4)拷贝/μL,表明qPCR方法的敏感性更强;该方法批内、批间重复性试验的变异系数分别小于1%和2%,重复性好。利用建立的qPCR方法和普通PCR方法对从呼和浩特市某赛鸽公棚随机采集的30份鸽肝脏样品进行PIAdV-I检测,结果显示前者的阳性检测率为63.3%(19/30),普通PCR方法的阳性率为36.7%(11/30),两种方法的阳性符合率均为100%。本研究首次建立PIAdV-I的q PCR检测方法,为鸽PIAdV-I的感染提供了敏感、快速的检测手段。
To develop a rapid detection method for pigeon adenovirus typeⅠ(PIAdV-Ⅰ),specific primers were designed according to the conserved region of PIAdV-ⅠFiber 2 gene.By optimizing the reaction conditions,a SYBR GreenⅠreal-time PCR(qPCR)was developed for rapid detection of PIAdV-I.The standard curve of SYBR GreenⅠq PCR showed that the linear relationship between Ct values and the standard plasmids in the range of 1×10^(3)copies/μL-1×10^(8)copies/μL was good,the correlation coefficient R~2was 0.995,and the amplification efficiency was 90%.The positive plasmid standard of PIAdV-II Fiber 2 gene,pigeon circovirus(PICV),Newcastle disease virus(NDV),Pasteurella multocida(PM)and avian pathogenic Escherichia coli(APEC)were all negative by this developed assay,which showed it had good specificity.In the sensitivity test,the detection limit of the recombinant plasmid standard p MD19-Fiber was 1×10^(2)copies/μL at the concentration of 1×10^(0)copies/μL to 1×10^(8)copies/μL and the detection limit of the common PCR was 1×10^(4)copies/μL,indicating that the fluorescence quantitative PCR method was more sensitive.The coefficient of variation(CV)of intra-batch and inter-batch assay was less than 1%and 2%,respectively,indicating good repeatability.Thirty samples of pigeon livers randomly collected from a racing pigeon shed in Hohhot were detected by SYBR Green I qPCR and the common PCR,the results showed that the positive rate of the former assay was 63.3%(19/30)and that of the common PCR was 36.7%(11/30).The positive coincidence rate of the two methods was 100%.In this study,a SYBR Green I q PCR assay for PIAdV-I was developed for the first time,which provided a sensitive and rapid method for PIAdV-I infection in pigeons.
作者
杨俊杰
韩晓语
刘春羽
王亚新
郭昊
温永俊
王凤雪
YANG Jun-jie;HAN Xiao-yu;LIU Chun-yu;WANG Ya-xin;GUO Hao;WEN Yong-jun;WANG Feng-xue(College of Veterinary Medicine,Inner Mongolia Agricultural University,Key Laboratory for Clinical Diagnosis and Treatment of Animal Diseases of Ministry of Agriculture and Rural Affairs,Hohhot 010018,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第10期1066-1070,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
内蒙古自治区自然科学基金项目(2020MS03047)
内蒙古农业大学高层次人才引进项目(NDGCC2016-22)。
