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

Samples that were positive by FCoV RT-qPCR, with a threshold cycle ≥ 36 (relative copy number ≤ 15), but that did not generate a 153 bp amplicon using primers F614/R766 were subjected to PCR to determine the presence of serotype 2 FCoVs. Primers were designed to amplify a ≈ 1820 bp fragment encompassing the S1 region of the S protein gene of serotype 2 FCoVs, as previously described [27] . Briefly, PCR was performed using: 2 × GoTaq Master Mix (Promega), 400 nM forward and reverse primers (FCoV S2 F1/FCoV S2 R3), 5 μL cDNA in a total volume of 25 μL; the following thermal profile: 95 °C for 2 min, 45 cycles of 95 °C for 15 s, 55 °C for 20 s and 72 °C for 2 min, followed by 72 °C for 5 min before being held at 10 °C; in an Agilent Thermal Cycler (Agilent Technologies). Positive and negative PCR controls were included in each reaction. Reaction products were separated by agarose gel electrophoresis to confirm that a single amplicon of the correct size was produced. One reaction product from each cat was subjected to a standard Sanger sequencing protocol using the amplification primers as sequencing primers.