Optimization and Validation of a Diagnostic Real-Time PCR for Specific Detection of Mycobacterium avium Subspecies Paratuberculosis

A diagnostic Real time PCR (qPCR) for detection of Mycobacterium avium subspecies paratuberculosis (MAP) was optimized using the ISMav2 sequence specific for MAP. The analytical sensitivity of the assay was 500 fg, and it was able to detect approximately 7 copies of the positive control plasmid construct. The qPCR detected up to 1x103 , 1x104 and 1x 105 MAP cells per reaction from spiked bovine semen, milk and feces respectively. The assay was reliable, reproducible and could be completed in 87 minutes. Comparative quantification for MAP copy number was established by utilizing normalized Cq values. The ISMav2 qPCR specifically detected 8 MAP strains, but was unable to amplify DNA from any other strains of Mycobacterium (n=5) or non-Mycobacterium strains (n=10). Several factors were tested to study their impact on the validation of the assay under field conditions. It was observed that sample size, number of sampling time points and test methods adopted for accepting the infection status were strongly correlated. The best return of validation estimates were obtained when sample-wise results of the qPCR were compared to the combined status of culture and acid fast bacilli (AFB) staining. The diagnostic sensitivity and specificity, positive and negative predictive values were estimated to be 100% (95% CI |63.06 -100.0|) and 99.9% (95% CI |99.44 100.0|), 88.89% (CI |51.75 -99.72|) and 100% (95%CI |99.75 -100.0|), respectively. Also, the qPCR and the MAP identification test conditions were strongly associated (κ = 0.941 (95% CI |0.825 – 1.00|). The validation estimates were true only when the sample size was large (n=1008) and sample collection was based on a single time point sampling strategy. Thus it appears that the assay could be considered as a reliable and rapid diagnostic test for field application.