Cfx96 real time fluorescence quantitative pcr apparatus

Viral DNA was extracted from HD11 cells or tissues by using the HiPure Tissue DNA Mini Kit (Magen, Guangzhou, China) according to the manufacturer’s instructions. The primers (forward: 5′-GGACCATCAACGGTGTTCAGG-3′ and reverse: 5′-GTCGCAGGATCGCTTCTTCGA-3′) for detecting the VP3 of CIAV were designed based on the sequence of the GD-101 strain of CIAV. The viral load of CIAV was detected by qPCR with SYBR Green qPCR Master Mix (Glpbio, Montclair, CA, USA). The samples were amplified and analyzed in a Bio-Rad Laboratories CFX96 real-time fluorescence quantitative PCR apparatus (Shanghai, China) with the following program: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 60 °C for 35 s. The results were analyzed using the 2^−∆∆CT method.

The HiPure Tissue DNA Mini Kit was purchased from Guangzhou Magen Bio & Tech Co., Ltd. (D3121, Guangzhou, China). The AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit was produced by Corning Incorporated (AP-MN-BF-VNA-250G, New York, United States). The freeze-dried powder for the real-time fluorescent RAA reaction was produced by Nanning Zhuangbo Bio & Tech Co., Ltd. (ZBA12001, Nanning, China). The EZ-press qPCR Kit was produced by Suzhou Yingze Biomedical Technology Co., Ltd. (EZB-qRT-R2, Suzhou, China). The pMD™19-T Vector Cloning Kit was produced by Takara Biomedical Technology (Beijing) Co., Ltd. (6,013, Beijing, China). This study used a Bio-Rad Laboratories CFX96 real-time fluorescence quantitative PCR apparatus (Shanghai, China).