2.5 Genomic RNA extraction and quantitative real-time PCR detection
Viral RNA was extracted from the embryo and spleen tissue using RNAprep pure Tissue Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. These extracted RNA samples were detected the concentration using DeNovix DS-11 Spectrophotometer (Nanodrop, USA) and stored at -20℃. The N-DRV nucleic acid in the sample were detected by the Taqman probe real-time quantitative PCR method previously established by our team, the N-DRV specific primers and TaqMan probe were designed to target the S2 gene of N-DRV strain, and the smallest detectable amount was 10 copies/μL. The primers and probe sequences were shown as follows: N-DRV-S1-F: 5’-ATGGATCGCAACGAGGTGATAC-3’ (958-978), N-DRV-R: 5’-CTAGCCCGTGGCGACGGT-3’ (1022-1042), Probe: 5’-FAM-AACGCCTGTGCACGAGCTGAAC-TAMRA-3’ (981-1022).
The qPCR amplification reaction is carried out with One Step PrimeScript™ RT-PCR Kit (TaKaRa, Dalian, China), and the reaction mixture containing 10μL of 2X One Step RT-PCR Buffer III, 0.4μL each of TaKaRa Ex Taq HS (5U/μl), PrimeScript RT enzyme Mix II, PCR Forward Primer (0.2 µmol/L) and PCR Reverse Primer (0.2 µmol/L), 0.4μL of Probe (0.2 µmol/L), 2μL of total RNA, and then add RNase Free dH2O in a final volume of 20μL. The qPCR was conducted with Applied Biosystems 7300FAST Real-Time PCR System under the following conditions: 42℃ for 5 min and 95 ℃ for 10 sec, 40 cycles denaturation at 95 ℃ for 5 sec and annealing and elongation at 60 ℃ for 20 sec, At the end of each annealing and elongation step, fluorescence signals for each sample were collected (Wang et al., 2020). The cycle threshold (CT) values and standard curve were analyzed by Sequence Detector software (version 2.1, Applied Biosystems).