Quantitative PCR Assay for C. pecorum Detection

Swab samples were stored at -20°C until the DNA was extracted as described by Devereaux et al. [13 (link)]. The extracted samples were screened for the presence of C. pecorum using a diagnostic quantitative real-time PCR (RT-PCR) targeting a 204 bp fragment of the chlamydial 16S rRNA gene. Assays were as described in Marsh et al. [14 (link)] except for the PCR mixture containing 1× QuantiTect SYBR Green PCR Master Mix (Qiagen) and 10 μM primers [14 (link)] made up to a final volume of 15 μl with PCR-grade water, as well as an increased initial denaturation to 15 mins at 94°C. All reactions were carried out on a Rotor-Gene Q 5-plex HRM platform (Qiagen).