Validation of IS900- qPCR assay to assess the presence of Mycobacterium avium subs. paratuberculosis in faecal samples according to the OIE procedure

Paratuberculosis is a chronic enteric progressive disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP). Despite cultural methods being considered the gold standard for the diagnosis of paratuberculosis, quantitative PCR (qPCR) assays have been developed for this purpose. These assays showed sensitivity and specificity comparable to cultural method but provide more rapid analysis results. Aim of our work was the validation of an IS900-qPCR assay for detection of MAP in faeces according to the OIE guidelines relative to the validation of assays for infectious diseases. The analytical and diagnostic characteristics and the reproducibility of the qPCR method were assessed. The robustness of the assay was evaluated using two extraction methods (silica column and magnetic beads DNA capture) and two qPCR systems (STEPONE™ and CFX96™). According to our validation, the analytical specificity, inclusivity and exclusivity were found to be appropriate for the use of this qPCR assay as a diagnostic test. Specifically, the limit of detection was approximately 100 CFU/g or even less if binomial approaches were used for the determination of the 95 % probability of detection (logit and clog-log models) with sufficient repeatability and reproducibility. Estimation of test accuracy was performed using a Bayesian two latent class model, in various scenarios combining different priors for prevalence and accuracy of the two tests used. All models were run considering three different cut-offs for qPCR. Our validation study underlines the good performance of this IS900-qPCR assay for diagnosis of MAP representing a valid and robust alternative to culture. Moreover, coupled with the semiautomatic magnetic beads DNA extraction method, this assay allows the rapid processing of numerous samples.