Virus nucleic acid kit

Clearly, 14 U of Turbo DNase (Ambion, Austin, TX, USA), 25 U of Benzonase Nuclease (Novagen, San Diego, CA, USA), 20 U of RNase I (Fermentas, Ontario, Canada), and 10 × DNase buffer (Ambion) were added to 127 μL of the supernatants to a final volume of 150 μL, followed by digestion at 37°C for 1 h. After removing free nucleic acid and eliminating the contaminating host genomic DNA, the viral nucleic acid in the obtained products was extracted using a Virus Nucleic Acid Kit (Bioer Technology, Hangzhou, China) according to the manufacturer's instructions. The total viral nucleic acids were reverse transcribed using anchored random primers and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). Anchored random primers (Table 2) were added separately to the viral nucleic acid and incubated at 75°C for 5 min and placed on ice for 5 min for denaturation. To acquire the reverse transcribed product, 40 U of RNase OUT, 200 U of Superscript III reverse transcriptase, 1 μL of 0.1 M dithiothreitol (DTT), 1 μL of 10 mM dNTPs, 4 μL of 5 × first strand buffer, and RNase-free H₂O were added to a final volume of 20 μL. It was incubated at 25°C for 10 min followed by 50°C for 60 min, and 75°C for 10 min.

In accordance with the alignment results of viral contigs and the match position of viral contigs with the corresponding viruses in GenBank, specific primers (Table 3) were designed and synthesized to identify the detected viruses. Viral nucleic acids were extracted using a Virus Nucleic Acid Kit (Bioer Technology) and amplified using the designed primers and a PCR Master Mix (Tiangen, Beijing, China).