An ISMap02-like insertion sequence in Mycobacterium spp. interferes with specific detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease or paratuberculosis (PTB), which is a chronic debilitating disease in ruminants, that is characterized by incurable enteritis and persistent diarrhea. ISMap02 is one of the major targets of PCR because it is present in multicopies (six copies) and known to be specific to MAP. However, in the present study, non-MAP mycobacteria were shown to be positive by ISMap02 targeting PCR. Two bacterial isolates (Sample ID: BO-038 and BO-042) were cultured from bovine fecal samples that produced positive results in three of two ISMap02 targeting PCR analyses with negative results in IS900 real-time PCR. Species identification using 16S rRNA gene sequencing and hsp65 gene partial sequencing revealed that strains BO-038 and BO-042 were M. virginiense and M. nonchromogenicum, respectively, which both belong to the M. terrae complex (MTC). Moreover, the two isolates shared a novel insertion sequence (IS) with high similarity to some parts of nucleotide sequences of ISMap02, and IS was presumed to be identical to that present in M. heraklionense. Both the novel IS and ISMap02 were characterized as IS1182 family members, and several sequences similar to ISMap02 were identified by BLAST analysis. In addition, the DDE transposase of the novel IS showed great similarity in the N-terminal portion with the IS5/1182 DDE transposase of other mycobacteria. These results suggest that ISMap02 has a conserved region with similarity to other ISs, and that the diagnostic value of the primer sets targeting that region should be re-addressed.

Park HT ，Park HE ，Jung YH ，Yoo HS ... -  《-》

Development of a nested PCR method targeting a unique multicopy element, ISMap02, for detection of Mycobacterium avium subsp. paratuberculosis in fecal samples.

Stabel JR ，Bannantine JP 《-》

Mycobacteria distenct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable IS900 polymerase chain reaction: implications for diagnosis.

PCR targeting the 5' end of IS 900 has been considered specific for identification of Mycobacterium avium subsp. paratuberculosis and is frequently applied to confirm the presence of this organism in the diagnosis of Johne's disease. IS 900 PCR has also been applied to studies of the aetiology of Crohn's disease. Mycobacterium spp. isolated from the faeces of 3 clinically normal animals in 2 Australian states appeared not to be M. paratuberculosis but were positive by IS 900 PCR. The isolates were characterized using mycobactin dependency, biochemical tests, IS 900 and 16 S rRNA sequencing and restriction fragment length polymorphism (RFLP) using IS 900 as probe. DNA sequencing confirmed that the isolates had between 71% and 79% homology with M. paratuberculosis in the region of IS 900 amplified, were most closely related to Mycobacterium scrofulaceum, and confirmed the usefulness of restriction enzyme analysis of amplified product to identify the false positive results. RFLP analysis with Bst Ell detected three to five copies of the IS 900 -like element in the isolates. These were located in molecular weight fragments that were clearly different to IS 900 in previously characterized strains of M. paratuberculosis. It is likely that these isolates are environmental mycobacteria. Southern blotting with an internal probe is unlikely to provide differentiation of M. paratuberculosis from these organisms. We recommend the adoption of restriction endonuclease analysis of IS 900 PCR product as a routine precaution to prevent the reporting of false positive IS 900 PCR results.

Cousins DV ，Whittington R ，Marsh I ，Masters A ，Evans RJ ，Kluver P ... -  《MOLECULAR AND CELLULAR PROBES》

Improved DNA Amplification of the Hallmark IS900 Element in Mycobacterium avium subsp. paratuberculosis: a Reexamination Based on Whole-Genome Sequence Analysis.

Bannantine JP ，Stabel JR ，Bayles DO ，Biet F ... -  《-》

Development and validation of a loop-mediated isothermal amplification assay-a rapid and sensitive detection tool for Mycobacterium avium subsp. paratuberculosis in small ruminants.

The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop-mediated isothermal amplification (LAMP) system, as a time-saving and user-friendly method in contrast to real-time PCR. For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10 fg μl-1 . After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698T , the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real-time PCR results. An internal amplification control was incorporated into the assay to exclude false-negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real-time PCR. Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended. This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies.

Sange MD ，Becker A ，Hassan AA ，Bülte M ，Ganter M ，Siebert U ，Abdulmawjood A ... -  《-》