A Novel Multiplex Real-Time PCR for the Identification of Mycobacteria Associated with Zoonotic Tuberculosis

Abstract

BackgroundTuberculosis (TB) is the leading cause of death worldwide from a single infectious agent. An ability to detect the Mycobacterium tuberculosis complex (MTC) in clinical material while simultaneously differentiating its members is considered important. This allows for the gathering of epidemiological information pertaining to the prevalence, transmission and geographical distribution of the MTC, including those MTC members associated with zoonotic TB infection in humans. Also differentiating between members of the MTC provides the clinician with inherent MTC specific drug susceptibility profiles to guide appropriate chemotherapy.Methodology/Principal FindingsThe aim of this study was to develop a multiplex real-time PCR assay using novel molecular targets to identify and differentiate between the phylogenetically closely related M. bovis, M. bovis BCG and M. caprae. The lpqT gene was explored for the collective identification of M. bovis, M. bovis BCG and M. caprae, the lepA gene was targeted for the specific identification of M. caprae and a Region of Difference 1 (RD1) assay was incorporated in the test to differentiate M. bovis BCG. The multiplex real-time PCR assay was evaluated on 133 bacterial strains and was determined to be 100% specific for the members of the MTC targeted.Conclusions/SignificanceThe multiplex real-time PCR assay developed in this study is the first assay described for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. Future validation of this multiplex assay should demonstrate its potential in the rapid and accurate diagnosis of TB caused by these three mycobacteria. Furthermore, the developed assay may be used in conjunction with a recently described multiplex real-time PCR assay for identification of the MTC and simultaneous differentiation of M. tuberculosis, M. canettii resulting in an ability to differentiate five of the eight members of the MTC.

Diagnosis of bovine tuberculosis: review of main techniques

“…However, it is limited as it does not distinguish M. canettii and M. pinnipedii. While SNP's have commonly been used for accurate discriminate of members of the M. tuberculosis Complex further validation of the SNP identified in this study for the specific detection of M. caprae is required (Reddington et al, 2011).…”

Bovine Tuberculosis in the Republic of Macedonia: Postmortem, Microbiological and Molecular Study in Slaughtered Reactor Cattle

“…In order to identify whether the RD4-isolates were all M. bovis or the Macedonian M. caprae isolates have deleted RD4, all of the RD4-isolates were subjected to the protocol for detection of M. caprae specific SNPs in the lepA gene (15,16). From the 79 (RD4-) isolates, 8 isolates were identified as M. caprae.…”

“…During this study, Domogalla et al (15) detected RD4-isolates of M. caprae. Considering this, the isolates were further identified by the presence of specific single nucleotide polimorfisms (SNPs) in the lepA gene by the previously described protocols (15,16).…”

Diagnóstico histopatológico e molecular de lesões sugestivas de tuberculose em búfalos abatidos nos municípios de Macapá e Santana, estado do Amapá

“…Fator este possivelmente relacionado ao volume e diluição de DNA empregados no referido estudo ou à quantidade de bacilos na amostra, uma vez que a sensibilidade de sistemas de PCR no diagnóstico de M. bovis encontra-se diretamente ligado a este fator (Collins 2011). Reddington et al (2011) relatam 100% de especificidade para PCR múltipla em tempo real (m-PCR) na identificação e diferenciação simultânea de micobactérias zoonó-ticas (M. bovis, M. bovis BCG e M. caprae) em bovinos. Shah et al (2002) também destacam a importância da utilização da m-PCR associada a técnicas microscópicas para identificação e diagnóstico diferencial da tuberculose.…”