Establishment and Application of Taq-Man Probe-based Multiplex qPCR Method for the Detection of DuCV,DEV and NGPV
[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.
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