2.2.2. Virus Quantitation by qPCR
For total viral counts for all samples, whole cell qPCR (quantitative Polymerase Chain Reaction) was performed using BCoV specific oligonucleotides and probes [22,23]. The BCoV sample was added to the qPCR SYBR Green reaction mixture (Applied Biosystems, Warrington, UK), and amplified in an automated thermocycler/analyzer (AB StepOne RT-PCR System, AB, Foster City, CA, USA) as described previously [24]. Broadly reactive primer pairs IN-2 (+) 5′ GGGTTGGGACTATCCTAAGTGTGA 3′ and IN-4 (–) 5′ TAACACACAACICCATCATCA 3′ were used to identify the bovine coronavirus (GenBank Access NC_003045). Dilutions of a plasmid containing the BCoV target sequence were used to create a standard calibration curve for each analysis and allow for the calculation of total viral gene copy number [25].