RESUMO
In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.
Assuntos
Brucella/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Técnicas Bacteriológicas/métodos , Brucella/genética , Brucelose/diagnóstico , Primers do DNA/genética , Humanos , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Fatores de TempoRESUMO
In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.
Assuntos
Animais , Humanos , Brucella/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas Bacteriológicas/métodos , Brucella/genética , Brucelose/diagnóstico , Primers do DNA/genética , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Fatores de TempoRESUMO
Tuberculosis in seals is caused by Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis complex. In this study, we evaluated the extent of genetic variability among Mycobacterium bovis and M. pinnipedii by microarray-based comparative genomics. We identified two deletions that are exclusive to M. pinnipedii: PiD1 that removes the orthologues of the M. tuberculosis genes Rv3530c and Rv3531c, and PiD2 that encompasses genes Rv1977 and Rv1978. Interestingly, a deletion overlapping the previously described RD2 region was identified in some isolates of Mycobacterium microti and further characterised.
Assuntos
Marcadores Genéticos , Genoma Bacteriano , Mycobacterium/genética , Otárias/microbiologia , Variação Genética , Mycobacterium/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade da EspécieRESUMO
Mycobacterium microti is the agent of tuberculosis in wild voles and has been used as a live vaccine against tuberculosis in man and cattle. To explore the M. microti genome in greater detail, we used a M. tuberculosis H37Rv genomic DNA microarray to detect gene deletions among M. microti isolates. A number of deletions were identified that correlated with those described previously (Infect. Immun. 70 (2002) 5568) but a novel M. microti deletion was also found (MiD4) which removes 5 genes that code for ESAT-6 family antigens and PE-PPE proteins. Southern blot experiments showed that this region was also deleted from M. pinnipedii, a mycobacterium isolated from seals that is closely related to M. microti. Genes encoding ESAT-6 antigens and PE-PPE proteins appear to be frequently deleted from M. microti, and the implications of this are discussed.
