Establishment and application of a TaqMan probe real-time fluorescence quantitative RT-PCR method for the detection of novel duck reovirus
1. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China; 2. Hebei Veterinary Biotechnology Innovation Center, Baoding 071001, China; 3. Beijing Institute of Animal Husbandry and Veterinary Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
In order to detect Novel duck reovirus (NDRV) rapidly and accurately, we designed specific primers and TaqMan probes to detect the NDRV σB gene, and optimized the reaction conditions. The test was conducted in the following ways: sensitivity testing, repeatability testing and clinical sample testing. The results showed that the standard curve equation of the TaqMan flfluorescence quantitative RT-PCR method was y=-3.354x+44.818, the amplification efficiency was 98.7%, and the correlation coefficient of the standard curve was 0.999; the method could specififically detect NDRV, but had no reaction signal for MDRV, ARV, TMUV and DHAV; the method detected plasmid standards. The lowest detection concentration was 10 copies/μL, which was 1,000 times more sensitive than the conventional PCR method; the intra-and inter-group coeffificients of variation of this method were both less than 2%; the results showed that the positive rate of NDRV was 45%, while the positive rate of conventional RT-PCR was 33%, which was more sensitive than the conventional RT-PCR. The results show that the TaqMan flfluorescence quantitative RT-PCR assay established in this study can provide reliable technical support for the monitoring and early diagnosis of NDRV.
2020, Vol. 43 
