Abi taqman fast virus 1 step master mix

Pan-Alphavirus assay was performed in a total volume of 10 μL including 2.5 μL of template, 2.5 μL of a 4X mix including selected primers and probe according to Table 3, 2.5 μL of molecular grade water and 2.5 μL of 4X ABI TaqMan® Fast Virus 1-Step Master Mix (Applied Biosystems, 850 Lincoln Centre Drive, Foster City CA94404, Cat #4444434). Oligonucleotides were purchased from Eurogentec®. TaqMan® hydrolysis probes were 5′ labeled using ATTO425 fluorochrome and 3′ quenched with BHQ1 (Black hole Quencher-1). Final concentration of primers and probes were respectively set at 500 μM and 250 μM. Cycling conditions were: 45 °C, 5 min, 98 °C, 20 s and 45 cycles comprising 2 steps, 98 °C, 3 s and 58 °C, 45 s with fluorescence reading using CYAN channel for detection of TaqMan® probe hydrolysis. RT-PCR cycling was set on a Roche LC480-II thermal cycler (Roche Applied Science, 68,298 Mannheim Germany, Cat #05015278001).

Eight-day-old ECEs were inoculated with 10² EID₅₀ of IBV H52 BI or recombinant IBV wild-type (rIBV-wt). Eggs were candled twice daily and 6, 12, 24, 36, and 48 hpi. Five previously selected eggs per virus strain were transferred to 4 °C for 16–24 h, and AF was aseptically harvested and stored at −80 °C. For analysis, AF samples were thawed and tenfold diluted in PBS without Ca and Mg, and nucleic acids were extracted with the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany) and the addition of carrier RNA, using the Hamilton Starlet pipet robot (Reno, Nevada, USA). Extracted nucleic acids were analyzed by RT-qPCR for the amount of IBV RNA following the protocol from Callison et al. [42 ] with small adaptations. Briefly, the same primers and probe were applied, while the thermoprofile was adapted for use of the ABI TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems by Thermo Fisher Scientific) and the Roche 480 LightCycler. All nucleic acid samples were run and analyzed in triplicates using a tenfold dilution series of IBV H52 BI as reference for quantification. The embryonic survival at each of the time points was calculated according to the number of embryos alive at each time point compared to the total number of animals still in the experiment. A paired t-test was performed to analyse the differences in embryonic death between IBV H52 BI and rIBV-wt.