The investigation of the truncated mbtA gene within the mycobactin cluster of Mycobacterium avium subspecies paratuberculosis as a novel diagnostic marker for real-time PCR

•The use of mbtA truncation as a novel target for MAP detection•This target displayed comparable detection results with the established f57 target.•The unique melting curve profile generated with this target is easily interpreted.•This target can be of great value when implemented in a multiplex qPCR assay.

The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP.Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34 pg and 104 CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 103 CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 103 CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 102 CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon.This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and with its unique melting curve profile adds increased specificity to MAP diagnostic tests.