title: Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and Regions of Difference-targeted dual-labelled hydrolysis probes.
abstract: Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of tuberculosis (TB) in both humans and animals. MTC species are genetically very similar but may differ in their epidemiology, namely geographic distribution and host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species of the MTC. In this work we describe a rapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals. The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110 element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows the assessment of the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows us to infer the species of the isolate as M. tuberculosis (if all RDs are present), Mycobacterium caprae (if only RD1 and RD4 are present) and Mycobacterium bovis (if only RD1 is present). The identification algorithm developed presented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970 (CI(P95%) 0.929-1.000). The assay is able to be adaptable to automation and implementation in the routine diagnostic framework of veterinary diagnostic laboratories, with a particular focus for reference laboratories.
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal; Grupo de Micobactérias, Unidade de Ensino e Investigação de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Costa
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Amaro
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal; Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Ana S
authors.initials: AS
authors.lastname: Ferreira
authors.affiliation: Grupo de Micobactérias, Unidade de Ensino e Investigação de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Machado
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal.
authors.forename: Teresa
authors.initials: T
authors.lastname: Albuquerque
authors.affiliation: Grupo de Micobactérias, Unidade de Ensino e Investigação de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal; Centro de Recursos Microbiológicos, CREM, Universidade Nova de Lisboa, Portugal.
authors.forename: Isabel
authors.initials: I
authors.lastname: Couto
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Botelho
authors.affiliation: Grupo de Micobactérias, Unidade de Ensino e Investigação de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal; Centro de Malária e Outras Doenças Tropicais, IHMT/UNL, Lisboa, Portugal.
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal; School of Pharmacy and Biomolecular Sciences, University of Brighton, United Kingdom. Electronic address: j.inacio@brighton.ac.uk.
authors.forename: João
authors.initials: J
authors.lastname: Inácio
mesh_terms: D000465:Algorithms; D000820:Animal Diseases / Q000175:diagnosis* / Q000382:microbiology*; D000818:Animals; D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014376:Tuberculosis / Q000662:veterinary*
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Bovine tuberculosis; IS6110; Multiplex real-time PCR; Mycobacterium bovis; Mycobacterium tuberculosis complex; Regions of Difference