title: Simultaneous detection and strain differentiation of Mycobacterium bovis directly from bovine tissue specimens by spoligotyping.
abstract: Culture of Mycobacterium bovis is used routinely to support field diagnosis of bovine tuberculosis; however, this method is slow. Rapid detection and strain-typing of M. bovis directly from 37 lesioned bovine lymph node specimens was performed by the polymerase chain reaction (PCR) based method, spoligotyping. Mycobacterial DNA was extracted from the specimens using a nucleic acid sequence capture technique. Two sets of specimens were tested, the first set comprising 16 decontaminated tissue homogenates from lesioned lymph node specimens which had been processed for BACTEC culture and a second set of 21 non-decontaminated lesioned lymph node specimens. Both sets of specimens had been frozen before analysis. Sequence capture PCR enabled detection and strain-typing of M. bovis directly from 15 of the 16 decontaminated homogenates and all 21 of the non-decontaminated tissues. Four spoligotype (ST) patterns were obtained from each set; ST1, ST2, ST3 and ST16 were detected in the decontaminated specimens and ST1, ST2, ST11 and ST14 in the non-decontaminated specimens. For both sets of specimens, ST1 was the predominant strain type detected. ST patterns obtained from the BACTEC cultures of the decontaminated specimens were in agreement with those obtained directly from the tissue. The sensitivity of detection by sequence capture-PCR compared very favourably with that of BACTEC culture. ST patterns were obtained directly from tissues of 34 of the 35 culture positive specimens and the two culture negative specimens. DNA extraction from the 21 non-decontaminated specimens involved an initial stomaching treatment. An assessment of sequence capture on both liquid alone and liquid and tissue homogenate combined, following stomaching, indicated that PCR was less successful on the liquid component alone.
authors.affiliation: Queen's University of Belfast, Stoney Road, Stormont, Belfast, UK.
authors.forename: S
authors.initials: S
authors.lastname: Roring
authors.forename: M S
authors.initials: MS
authors.lastname: Hughes
authors.forename: R A
authors.initials: RA
authors.lastname: Skuce
authors.forename: S D
authors.initials: SD
authors.lastname: Neill
mesh_terms: D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D008198:Lymph Nodes / Q000382:microbiology; D008863:Microspheres; D009163:Mycobacterium bovis / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D010865:Pilot Projects; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D014380:Tuberculosis, Bovine / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial