Chamq sybr qpcr mix

Specific primers for qPCR detection were designed and synthesized for the ASFV p72 gene (GenBank: mk333180.1) and CSFV NS5B gene (GenBank: ay775178.2) (Supplementary Table S2), with amplified target gene lengths of 80 bp and 174 bp, respectively. The qPCR reaction system is described as below: A 20 µL volume of qPCR amplification reaction consisted of 10 µL of ChamQ SYBR qPCR Mix (Vazyme Biotech Co., Ltd., China), 0.4 µL of each of the primers (10 µM), 8.2 µL of ultrapure water and 1 µL of DNA template. The qPCR programs were as follows: Pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, and extension at 60°C for 30 s, with a total of 40 cycles set up.

After RNA extraction, RNA quality and quantity were measured using a NanoDropTM²⁰⁰⁰ (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HiScript RT Kit (R222, Vazyme, Nanjing, China) was used to reverse the total RNA into cDNA. qRT-PCR was performed on a Step-One-Plus Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific., Waltham, MA, USA) using ChamQ SYBR qPCR Mix (Q711, Vazyme, Nanjing, China). The PCR primers are shown in Supplementary Table S2, and qRT-PCR was performed as described previously [14 (link)]. Each assay was performed more than 3 times. The expression level was calculated with the 2^−ΔΔCt method and normalized to the Actb expression level, defined as 1.0.