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 then underwent conventional PCR to amplify a 153 base-pair (bp) DNA fragment encompassing amino acid positions M1058L and S1060A in the S protein gene of serotype 1 FCoV, and subsequent pyrosequencing of the amplicon. Amplification and sequencing primers (Table 1) were designed using a combination of PyroMark assay design software (Qiagen), Primer3 [25] and MFold [26] , and were made by Eurofins (MWG Operon) or Metabion. Degeneracies were added to the primers, and the location of the primers optimised, based upon a sequence alignment comprised of all available serotype 1 FCoV genomes (data not shown). Briefly, PCR was performed using: 2 × GoTaq Master Mix (Promega), 200 nM forward and biotinylated reverse primers (F614/R766), 5 μL cDNA in a total volume of 25 μL; the following thermal profile: 95 °C for 2 min, 40 cycles of 95 °C for 15 s, 52 °C for 20 s and 72 °C for 20 s, before being held at 10 °C; in a MJ Mini Gradient Thermal Cycler. Samples that failed to produce definitive sequence data were pyrosequenced following repeat amplification using the same PCR protocol with 50 cycles of amplification.