基于Taq-Man探针的多重qPCR检测DuCV·DEV·NGPV方法的建立与应用
Establishment and Application of Taq-Man Probe-based Multiplex qPCR Method for the Detection of DuCV,DEV and NGPV
杨孟豪 1李丽湲 2肖雅清 3刘霞 1刘永夏 4孟凯2
作者信息
- 1. 山东省农业科学院家禽研究所,山东济南 250100;山东农业大学动物科技学院,山东泰安 271000
- 2. 山东省农业科学院家禽研究所,山东济南 250100
- 3. 山东省农业科学院家禽研究所,山东济南 250100;河北农业大学,河北保定 071000
- 4. 山东农业大学动物科技学院,山东泰安 271000
- 折叠
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、NG-PV,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2.5×101 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.
Abstract
[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 con-structed,the reaction conditions were optimized,and multiplex quantitative PCR detection methods for DuCV,DEV and NGPV were estab-lished.[Result]The method could identify DuCV,DEV and NGPV at the same time,and did not cross-react with other common duck virus dis-eases.The minimum detection amount of DuCV,DEV and NGPV was 2.5×101 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 detec-tion method,the positive coincidence rate was 100%.[Conclusion]A multiplex real-time PCR method based on Taq-Man probes was estab-lished to detect DuCV,DEV,NGPV simultaneously,quickly and quantitatively.It is more sensitive and practical than ordinary PCR,which ef-fectively 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.
关键词
鸭圆环病毒/鸭肠炎病毒/新型鹅细小病毒/Taq-Man探针/荧光定量PCRKey words
Duck circovirus(DuCV)/Duck enteritis virus(DEV)/Novel goose parvovirus(NGPV)/Taq-Man probes/RT-PCR引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据