Viral RNA Detection by RT-qPCR

The total RNA was extracted using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) following the protocol of the manufacturer after tissues were ground by a freeze-grinding machine (60 MHz, 2 min) (HODER Beijing N. 9548R). One-step nucleic acid detection kit (DaAn Gene, Guangzhou, China) was used to detect the RNA genome, which was determined by absolute RT-qPCR on a 7500 Real-time Quantitative PCR System (Applied Biosystems, Foster City, USA) under the following program, namely, 1 cycle at 50°C for 15 min, 95°C for 15 min; 40 cycles at 94°C for 15 s, and 55°C for 45 s. The number of viral RNA copy was calculated by generating a standard curve as previously described (Li et al., 2017 (link)). Viral RNA copy number was expressed as log₁₀ RNA copies per μl.