Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis

Barker, Emily N.; Stranieri, Angelica; Helps, Chris R.; Porter, Emily L.; Davidson, Andrew D.; Day, Michael J.; Knowles, Toby; Kipar, Anja; Tasker, Séverine

Quantitative PCR was performed using: 2 × GoTaq Master Mix (Promega), 200 nM forward and reverse primers (P009/P010), 25 nM TaqMan probe (P1), 2.5 mM MgCl2 and 5 μL cDNA in a total volume of 25 μL; the following thermal profile: 95 °C for 2 min and 40 cycles of 95 °C for 15 s, 55 °C for 15 s and 72 °C for 15 s; in an Agilent Mx3005P qPCR System (Agilent Technologies). The primers and probe were synthesized by Metabion (Metabion International) and were described previously [24] . Fluorescence was detected at 520 nm during the extension phase. FCoV cDNA was used as a positive control and RNase-free water as a negative control. Relative FCoV copy number per reaction was calculated for positive samples, as previously described [20] .