Rotor gene probe rt pcr kit

To detect and quantify influenza A virus on the collector plates as well as in guinea pig tissue samples, we used a One-Step Taq Man real-time RT-PCR assay46 (link) with primers F1-mxA (150 nM) (5´-AAGACCAATYCTGTCACCTCTGA-3´), F3-mxA (150 nM) (5´-CAAGACCAATCTTGTCACCTCT GAC-3´) and R1-mxA (900 nM) (5´-TCCTCGCTCACTGGGCA - 3´) and probes P1-Mx (110 nM) (5´-FAM-TTGTGTTCACGCTCACC–MGB–3´) and P2-Mx (110 nM) (5´-FAM-TTTGTATTCACGCTCACCG–MGB -3´), with the Rotor-Gene Probe RT-PCR Kit (Qiagen). The real-time PCR reaction was performed in a Corbett Rotor-Gene 6000 (Qiagen) with the following cycling protocol: 50 °C for 10 min, followed by 45 cycles of 95 °C for 5 seconds and 57 °C for 15 seconds.

Viral RNA was extracted (EZ1 Advanced XL-QIAGEN; Hilden, Germany) and analysed using a quantitative one-step real-time RT-PCR with primers and probes targeting the S segment. The Rotor-Gene Probe RT-PCR kit (QIAGEN, Hilden, Germany) and Rotor-Gene Q platform were used. Viral load was measured with the intra-assay standards as described before [19 ].
The presence of IgG and IgM antibodies against CCHFV in serum was detected using an indirect enzyme-linked immunosorbent assay (ELISA) (Vector-Best, Novosibirsk, Russia). ELISA tests were completed in two sessions. According to the manufacturer’s instructions, optical densities (OD) > 0.214 and > 0.250 for IgM and IgG, respectively, were considered as positive results in the first session and > 0,214 and > 0,240 for the second. Higher OD values that could not be read by the ELISA reader were taken as 3.0. All tests were performed at the virology laboratory of the Public Health Institute of Turkey.