Plasmid mini kit 2

Viral RNA samples were extracted using Trizol reagent (Axygen, China), according to the manufacturer’s instructions. RNA was eluted into RNAse-free ddH₂O and stored at − 80 °C until use. In this study, reverse transcription reactions of extracted RNA were performed with a PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China).
The target fragment was obtained by amplification of cDNA with conventional primer and cloned into a pMD19-T vector (Takara, Dalian, China). The recombinant vector was transformed into DH5α Escherichia coli cells and shaken overnight at 37 °C; the cells were then purified with plasmid Mini Kit2 (Omega BioTek, USA), according to the manufacturer’s recommendations (Takara, Dalian, China). The positive recombinant plasmids were confirmed through sequencing analysis (Takara, Dalian, China). The recombinant plasmid concentrations were measured with UV spectrophotometry (NanoDrop one) and converted into copy numbers. The plasmid DNA was stored at − 80 °C until further use.

All target gene fragments were synthesized and constructed into pMD19-T vector by BGI (Shenzhen, China), then were transformed into DH5α Escherichia coli cells following the manufacture's recommendations (TaKaRa, Dalian, China) and purified by Plasmid Mini Kit 2 (Omega Bio-tek, USA). The standard plasmids were tested and verified, and sent to BGI (Shenzhen, China) for sequencing. The concentration of the reconstructed standard plasmids was tested by a Qubit Fluorometer.