Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and regions of difference-targeted dual-labelled hydrolysis probes

Pedro Costa, Ana Amaro, Ana S. Ferreira, Diana Machado, Teresa Albuquerque, Isabel Couto, Ana Botelho, Miguel Viveiros, João Inácio

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

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 IS. 6110 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.

Original languageEnglish
Pages (from-to)13-22
Number of pages10
JournalJournal Of Microbiological Methods
Volume107
DOIs
Publication statusPublished - Dec 2014

Keywords

  • Bovine tuberculosis
  • IS6110
  • Multiplex real-time PCR
  • Mycobacterium bovis
  • Mycobacterium tuberculosis complex
  • Regions of Difference

UN Sustainable Development Goals (SDGs)

  • SDG 3 - Good Health and Well-Being

Fingerprint Dive into the research topics of 'Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and regions of difference-targeted dual-labelled hydrolysis probes'. Together they form a unique fingerprint.

Cite this