基于 Taq-Man 探针的多重 qPCR检测 DuCV DEVNGPV 方法的建立 与应用.

[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 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 2.5×10¹ 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 detection 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. [ABSTRACT FROM AUTHOR]