摘要
[目的]建立一种快速、特异的鸭圆环病毒(DuCV)、鸭肠炎病毒(DEV)、新型鹅细小病毒(NGPV)的多重荧光定量PCR方法。[方法]针对DuCV、DEV、NGPV的基因保守区域序列,设计合成3对特异性引物及3种荧光基团标记的Taq-Man探针。构建重组质粒标准品,优化反应条件。建立针对DuCV、DEV、NGPV的多重荧光定量PCR检测方法。[结果]该方法能同时鉴别检测DuCV、DEV、NGPV,并且与其他常见鸭病毒病不发生交叉反应。对DuCV、DEV、NGPV最低检出限均为2.5×10^(1)copies/μL。该方法重复性好,组间和组内试验变异系数低于1.0%。用该方法检测150份临床样本,结果显示,DuCV、DEV、NGPV的阳性率分别为32.0%(48份)、23.3%(35份)和18.7%(28份),DuCV和DEV混合感染率为4.6%(7份),DuCV和NGPV混合感染率为8.6%(13份),总阳性率为87.3%(131份),未检出DEV和NGPV混合感染的结果。与常规PCR检测方法比较,阳性符合率为100%。[结论]该研究建立的基于Taq-Man探针的多重荧光定量PCR方法能够同时、快速、定量检测DuCV、DEV、NGPV。较普通PCR更敏感,实用性更强,有效避免了多次检测耗时长、费用高的问题,为DuCV、DEV、NGPV的监测和防控提供了有效可靠的检测技术。
[Objective]To establish a rapid and specific multiplex fluorescence quantitative PCR method for duck circovirus(DuCV),duck enteritis virus(DEV)and novel duck parvovirus(NGPV).[Method]Three pairs of specific primers and three fluorophore-labeled Taq-Man probes were designed and synthesized for the conserved region sequences of DuCV,DEV and NGPV.Recombinant plasmid standards were constructed,the reaction conditions were optimized,and multiplex quantitative PCR detection methods for DuCV,DEV and NGPV were established.[Result]The method could identify DuCV,DEV and NGPV at the same time,and did not cross-react with other common duck virus diseases.The minimum detection amount of DuCV,DEV and NGPV was 2.5×10^(1)copies/μL.The method was reproducible,and the coefficient of variation between and within groups was less than 1.0%.A total of 150 clinical samples were tested with this method,the results showed that the positive rates of DuCV,DEV and NGPV were 32%(48 cases),23.3%(35 cases)and 18.7%(28 cases),respectively,the mixed infection rates of DuCV and DEV were 4.6%(7 cases),the mixed infection rate of DuCV and NGPV was 8.6%(13 cases),and the total positive rate was 87.3%(131 cases),and the results of mixed infection of DEV and NGPV were not detected.Compared with the conventional PCR detection method,the positive coincidence rate was 100%.[Conclusion]A multiplex real-time PCR method based on Taq-Man probes was established to detect DuCV,DEV,NGPV simultaneously,quickly and quantitatively.It is more sensitive and practical than ordinary PCR,which effectively avoids the problems of time-consuming and cost-effective multiple tests,and provides an effective and reliable detection technology for the monitoring and prevention of DuCV,DEV and NGPV.
作者
杨孟豪
李丽湲
肖雅清
刘霞
刘永夏
孟凯
YANG Meng-hao;LI Li-yuan;XIAO Ya-qing(Poultry Research Institute of Shandong Academy of Agricultural Sciences,Jinan,Shandong 250100;College of Animal Science and Technology,Shandong Agricultural University,Tai’an,Shandong 271000;Hebei Agricultural University,Baoding,Hebei 071000)
出处
《安徽农业科学》
CAS
2024年第22期178-182,共5页
Journal of Anhui Agricultural Sciences
基金
山东省自然科学基金项目(ZR2021MC060)。
