First molecular detection of mycobacterium bovis in environmental samples from a French region with endemic bovine tuberculosis

International audienceAimsThe aim of the study was to determine the prevalence of Mycobacterium bovis (the causative agent of bovine tuberculosis, bTB) in environmental matrices within a French region (Cote d'Or) affected by this zoonotic disease. Methods and ResultsWe report here the development and the use of molecular detection assays based on qPCR (double fluorescent dye-labelled probe) to monitor the occurrence of Mycobacterium tuberculosis complex (MTBC) or Myco.bovis in environmental samples collected in pastures where infected cattle and wildlife had been reported. Three qPCR assays targeting members of the MTBC (IS1561 and Rv3866 loci) or Myco.bovis (RD4 locus) were developed or refined from existing assays. These tools were validated using Myco.bovis spiked soil, water and faeces samples. Environmental samples were detected positive for the presence of MTBC strains and Myco.bovis in the environment of bTB-infected farms in the Cote d'Or region. ConclusionsThe development of molecular assays permitted testing of several types of environmental samples including spring water, sediment samples and soils from badger setts entrance located in the vicinity of these farms, which were repeatedly contaminated with Myco.bovis (up to 87x10(3) gene copies per gram of badger sett soil). For the first time, direct spoligotyping of soil DNA enabled identification of Myco.bovis genotypes from environmental matrices. Significance and Impact of the StudyAll together, these results suggest that Myco.bovis occurs at low levels in environmental matrices in Cote d'Or within the bTB-infected area. Drinking contaminated water or inhaling contaminated bioaerosols might explain cattle infection in some cases

Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes

WOS: 000353219700015In this report, we present a simple electrochemical detection protocol for the detection of specific PCR-amplified DNA fragments, based on incorporation of biotin tags into DNA amplicons during PCR run in the presence of a biotinylated nucleoside triphosphate. For detection, an enzyme-linked electrochemical system involving streptavidin-alkaline phosphatase conjugate attached to the biotinylated DNA, adsorbed at the surface of a disposable pencil graphite electrode, is used. The enzyme converts an inactive indicator, 1-naphthyl phosphate, into electrochemically oxidizable indicator 1-naphthol that is subsequently detected. Excellent selectivity of this fast, facile, and inexpensive analysis not requiring any sophisticated electrode modification and its applicability for off-line monitoring of DNA amplification is demonstrated. Applications of the technique include detection of the presence of specific nucleotide sequences in biological samples, such as sequences related to pathogenic microorganism or transgenes. [GRAPHICS] .Czech Science FoundationGrant Agency of the Czech Republic [P206/11/1638, P206/11/P739]; ASCRCzech Academy of Sciences [RVO 68081707]; Turkish Scientific and Technological Research CouncilTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111T050]; ASCR (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111T050]This work was supported by the Czech Science Foundation (Grant P206/11/1638 to M. F. and P206/11/P739 to P. H.) and by the ASCR (RVO 68081707). A. E and M. F acknowledges to the grant of international joint project through between Turkish Scientific and Technological Research Council and the ASCR (TUBITAK Project No. 111T050)