Comparison and Validation of IS900-Sequence and Putative Sequences as Specific Target to Detect Mycobacterium avium subsp. paratuberculosis

Polymerase chain reaction (PCR) has been used to improve the identification of Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to compare and validate real-time PCR results of IS900-insertion sequence and nine putative sequences as diagnostic templates to detect MAP. MAP reference strains, field isolates, and non-MAP species were included in the study to validate the specificity of the target genes by PCR detection. All the IS900 insertion sequences and the putative sequences were specifically amplified from the field isolates and reference MAP strains whereas no amplifications were found from the six non-MAP strains. Uniplex and multiplex-PCR of IS900 and putative sequences showed the presence of two distinct amplicons respectively. Furthermore, in silico PCR simulation showed that the primers used in this study for IS900 sequences and the putative sequences amplified only from known MAP and were negative for 63 non-MAP species. Early, accurate and precise diagnostic protocol will assist to track contaminated herds, food products and would ultimately contribute to the control and prevention of Johne's disease. Based on our results within the size of our samples, the IS900 insertion sequence and the repetitive sequences were amplified only in MAP. The insertion/repetitive sequences targeted in the PCR are not found in the non-MAP species tested in the experiment. Furthermore, the in silico PCR amplification supported the in vivo PCR results.