Capripox Real-Time PCR Assay for Detection

Total DNA was extracted from skin lesions (scabs or nodules), blood, saliva, nasal swabs, fecal, and milk samples using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). DNA was then tested using a capripox generic real-time PCR assay using a previously described primer and probe mix [36 (link),37 (link)] that amplified a part of the P32 envelope protein gene. The qPCR reaction was prepared in a total volume of 12.5 µL consisting of 6 µL of Luna^® Universal Probe qPCR Master Mix (NEB, Hitchin, UK), 2 µL of primer–probe mix, 2 µL of nuclease-free water, and 2.5 µL of template DNA. The PCR was carried out on an ABI 7500 Fast thermal cycler (Applied Biosystems, Waltham, MA, USA) with a cycle condition of one cycle at 95 °C for 2 min (activation) followed by 40 cycles at 95 °C for 15 s (denaturation) and 60 °C for 30 s (annealing and extension). The cycle threshold (Ct) values obtained at ≤35 from the clinical samples were considered positive.