Taqman ez rt pcr core reagents kit

RNA isolation and RT-PCR were performed as described previously (14 (link), 15 (link)). Briefly, RNA from swabs and tissue suspensions was isolated by using a MagNaPure LC system with the MagNaPure LC total nucleic acid isolation kit (Roche Diagnostics, Almere, the Netherlands). Real-time RT-PCR assays were performed on an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) by using the TaqMan EZ RT-PCR core reagents kit (Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands) according to the manufacturer’s instructions. For each run, the samples were prepared and processed in parallel with several negative and positive control samples. Virus titers were determined by serial 10-fold dilution of the homogenized tissue samples and swabs on MDCK cells, as described elsewhere (14 (link), 15 (link)). Virus titrations were performed in triplicate.

Sterile cotton swabs were used to collect cloaca, throat or combined cloaca-throat samples from each individual goose. Samples were stored in transport medium at 4°C for maximally two weeks until transported to the laboratory, where samples were stored at -80°C until testing [18 ]. RNA was isolated by the MagnaPure LC system with the Magna-Pure LC total nucleic acid isolation kit (Roche Diagnostics, Almere, the Netherlands), and the AIVs were detected by a generic real-time reverse transcriptase PCR (RRT-PCR) assay targeting the matrix (M) gene (M RRT-PCR). Amplification and detection were performed on an ABI 7700 machine with a TaqMan EZ RT-PCR core reagents kit (Applied Biosystems, Nieuwerkerk aan den IJssel, the Netherlands) and 20 μl of RNA eluate in an end volume of 50 μl. A more detailed method description can be found in previous studies [18 ,27 (link)].

Cloacal and oropharyngeal swabs were analyzed separately for the presence of influenza A virus. For full details on RNA isolation and virus detection, see Munster et al. (2009 (link)). In short, RNA was isolated using a MagNA Pure LC System (no. 12236931001) with the MagNA Pure LC Total Nucleic Acid Isolation Kit (no. 0303850500; Roche Diagnostics, Almere, the Netherlands). Influenza A virus was detected using a generic real-time reverse transcriptase PCR assay targeting the matrix gene (M RRT-PCR). Amplification and detection were performed on an ABI 7700 machine with a TaqMan EZ RT-PCR core reagents kit (no. N808-0236; Applied Biosystems, Nieuwerkerk aan den IJssel, the Netherlands). Samples were considered positive for influenza A virus if the cycle threshold (C_T) value, which is the first real-time amplification cycle in which matrix gene amplification was detected, was ≤40. The C_T-value is inversely proportional to the number of virus particles in a sample.