Setting up a PCR for the detection of Bovine Papilloma Virus in equine sarcoids
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Introduction. The Bovine Papilloma Virus (BPV) types 1 and 2 are thought to play a causal role in sarcoid pathogenesis. To support a clinical diagnosis of a sarcoid lesion PCR for detection of BPV DNA is performed. Aim of the study. i) the set-up and validation of a PCR to detect BPV DNA in skin tissue samples and swabs derived from equine sarcoids ii) analysis of retrospective data to evaluate diagnostic value of the BPV PCR with respect to histologic examination. Materials and methods. Primer- and probe concentrations were optimized using primer- and probe matrices. Two different DNA extraction methods were compared. Fifty-six samples (10 swabs and 46 skin tissue samples) were tested with the newly set-up PCR. Outcomes were compared with results from a reference laboratory (for 23 samples), histological outcomes (for 11 samples) or presumptions of BPV absence (for 22 healthy skin samples). Retrospective data was analyzed by classifying patients based on histologic and PCR test outcomes. Relative sensitivity, relative specificity, overall agreement and Cohen’s kappa were calculated. Results. The 300 nM primer concentration and 50 nM probe concentration were identified as optimal concentrations. The DNeasy® Blood & Tissue kit (Qiagen) was the least labor intensive method for DNA extraction of biopsies. The High Pure PCR Template Preparation kit (Roche diagnostics) yielded significantly lower Cp values for DNA extraction of swabs (p=0.016). For 22/23 samples outcomes of the newly set-up PCR agreed with the outcomes of the reference laboratory. For 8/11 samples outcomes of the newly set-up PCR agreed with histologic outcomes. In all 22 healthy skin samples no BPV was detected with the newly set-up PCR. Relative sensitivity of the PCR compared to histologic examination was 98.4%, relative specificity was 68.2%. Overall agreement was 90.4%, corresponding with a Cohen’s kappa score of 0.729 (95% CI 0.518 – 0.940). Conclusion. Validation of the PCR was successful for BPV detection in equine skin tissue samples. The PCR can be applied as diagnostic tool, though PCR outcomes must always be interpreted in combination with clinical data. Validation of the PCR for BPV detection in swabs was not yet finished.