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).