title: 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.
abstract: 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 0.34pg and 104CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 103CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 103CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 102CFU/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.
authors.affiliation: Cork Institute of Technology, Department of Biological Sciences, Rossa Avenue, Bishopstown, Cork, Ireland.
authors.forename: Marcel
authors.initials: M
authors.lastname: de Kruijf
authors.affiliation: Cork Institute of Technology, Department of Biological Sciences, Rossa Avenue, Bishopstown, Cork, Ireland.
authors.forename: Aidan
authors.initials: A
authors.lastname: Coffey
authors.affiliation: Cork Institute of Technology, Department of Biological Sciences, Rossa Avenue, Bishopstown, Cork, Ireland. Electronic address: jim.omahony@cit.ie.
authors.forename: Jim
authors.initials: J
authors.lastname: O'Mahony
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001482:Base Composition; D015415:Biomarkers / Q000032:analysis*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005243:Feces / Q000382:microbiology; D008025:Ligases / Q000235:genetics*; D057230:Limit of Detection; D009161:Mycobacterium / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D016927:Mycobacterium avium subsp. paratuberculosis / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D010080:Oxazoles / Q000737:chemistry*; D010283:Paratuberculosis / Q000175:diagnosis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D015415:Biomarkers; D017931:DNA Primers; D004269:DNA, Bacterial; D010080:Oxazoles; C018608:mycobactins; D008025:Ligases; C526815:salicyl-AMP ligase, Mycobacterium tuberculosis
keywords: Johne's disease; Mycobacterium avium subspecies paratuberculosis; Mycobactin; Real-time PCR; f57