Titan one rt pcr kit

DVGs from cell cultured purified virions and from infected mice lung tissues were amplified by RT-PCR (Titan-One RT-PCR kit, Roche) as indicated above. Obtained products were amplified with Taq Polymerase (Sigma) for further cloning into pGEM-T vector using pGEM-T Easy kit (Promega). Selected clones were sequenced by Sanger method and obtained sequences were analyzed to confirm that they corresponded to defective genomes, including the 3′and 5′ends and a large internal deletion of the full-length viral segment.

RT-PCR for the PA or PB2 segments was used to determine the presence of DVGs and their relative amount to the full-length RNA of the same viral segment. To detect full-length segments, internal primers were used to amplify a central fragment, which is not present in DVGs. To detect DVGs, the same RNA sample and external primers of the PA segment were used in a separate reaction. Short amplification times were applied for the detection of both, internal fragment corresponding to full-length segment and DVGs, to allow detection of RNAs up to 1000nt in length. The method is illustrated in Fig 2A. The reverse transcription reaction was performed for 30 min (42°C), followed by PCR (35 rounds at 94°C for 30 s, 53°/58°C for 40 s, and 68°C for 40 sec using the Titan-One RT-PCR kit (Roche)). As a specificity control, the primers and RT-PCR conditions for DVGs amplification were used with a plasmid encoding the full-length PA segment, and no amplification product was obtained (S1A Fig). Additionally, primers and amplification conditions for internal fragment corresponding to full-length segment were used with purified DVGs, and no amplification product was obtained (S1A Fig).