Rapure viral rna dna kit

The RaPure viral RNA/DNA Kit (Magen, Guangzhou, China) was used to extract nucleic acids from clinical samples. The extracted total nucleic acids were used for RNA reverse transcription (RT) using HiScriptⅡ1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and amplification. The reaction system volume of RT was 20 µL, consisting of 10 µL of 2 × RT Mix, 1 µL of random hexamers, 7 µL of total RNA and 2 µL of HiScript Ⅱ Enzyme Mix. Finally, the cDNA was stored at −20 °C.
The reaction system volume of mPCR for PAIT and PAFR was 20 µL, including 10 µL of 2 × Taq Master Mix (Vazyme, Nanjing, China), 1 µL of template, 1 µL of the forward primer (10 mM), 1 µL of reverse primer (10 mM) and 7 µL of ddH₂O. The single PCR program was as follows: 98 °C for 3 min; 36 cycles of 98 °C for 20 s, 57 °C for 1 min, and 72 °C for 1 min 30 s; and 72 °C for 5 min. The products of PCR were detected with 2% agarose gel electrophoresis. The PCR amplification fragments of FCV (1940 bp), FHV-1 (1014 bp), FeLV (608 bp), C. felis (500 bp), IAV (155 bp), FCoV (726 bp), FeAstV (418 bp), FPV (337 bp) and FeKoV (198 bp) were ligated into the pMD 18-T vector (TaKaRa, Dalian, China). The ligation products were transformed and cultured, and then the positive plasmids were extracted. Additionally, the positive test plasmids were sent to the Beijing Genomics Institute (Guangzhou, China) for sequencing.

Total nucleic acids (TNAs) from the positive controls and twenty nasal swabs (NS) and 20 anal swabs (AS) for clinical samples were extracted using the RaPure Viral RNA/DNA Kit (Magen, Guangzhou, China) according to the manufacturer’s protocol. The extracted TNA was divided into two parts; one was used for the amplification of the DNA viruses (CAV-2, CanineCV and CPV) and the other for the reverse transcription (RT) and amplification of the RNA viruses (CDV, CIV, CPIV and CCoV). The RNA extracted from the positive controls and clinical samples was reverse transcribed to cDNA using random primer (TaKaRa, Dalian, China) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The cDNA and DNA were stored at -20°C until PCR amplification.

According to the manufacturer’s instructions, viral nucleic acids were extracted using the RaPure Viral RNA/DNA Kit (Magen, China), and F81 DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen, China). The reverse transcription reaction was performed to synthesize first-strand cDNA following the manufacturer’s instructions (Vazyme, China). Eventually, the samples were stored in a -80 ℃ freezer.