Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex

Abstract

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

Mycobacterium tuberculosis, which belongs to Mycobacterium tuberculosis Complex (MTC) together with M. bovis, M. bovis BCG, M. africanun, M. microti, M.canettii and M. pinnipedii, is the most common etiological agent of human tuberculosis (TB). However M. bovis may also be a relevant human pathogen in countries with high prevalence of bovine TB (OIE, 2009). Immuno-suppressing diseases, especially caused by Human Immunodeficiency Virus (HIV), is a risk factor for the development of human TB by M. bovis (Thoen et al., 2006; Cícero et al., 2009). Since M. bovis-caused human TB is clinically, pathologically and radiologically undistinguishable from M. tuberculosis-caused TB (De La Rua-Domenech, 2006), the fast differentiation of the two species has a high epidemiologic importance (Leite et al., 2003). Among molecular methods for detecting species belonging to the MTC, several markers have been employed. The IS6110 is highly used since this marker is found in several copies in the MTC genome (Eisenach et al., 1990; Ogusku et al., 2004). The pncA gene, which encodes the pyrazinamidase enzyme (PZAase) that converts the pro-drug pyrazinamide (PZA) to pyrazinoic acid (POA), has a single point mutation at nucleotide 169C > G (appears to be unique to M. bovis) is one of the markers that is commonly used to distinguish M. bovis from others MTC species (De Los Monteros et al., 1998; Kidane et al., 2002). In the current study we evaluated a rapid test to differentiate M. bovis from other MTC species by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. DNA from M. tuberculosis H₃₇Rv (ATCC 27294), M. bovis AN5, M. avium (Central Laboratory - LACEN/PR), M. smegmatis (LACEN/PR), M. kansasii (LACEN/PR), M. fortuitum (LACEN/PR), M. szulgai (LACEN/PR), M. massilienses (LACEN/PR), M. abcessus (LACEN/PR), M. chelonae (LACEN/PR) reference strains, one M. bovis BCG, 17 M. bovis, isolated from bovine lymph nodes and indentified by biochemical and molecular methods in a previous work (Cardoso et al., 2009), 30 M. tuberculosis clinical isolates indentified by biochemical and molecular methods in a previous work (Noguti et al., 2010) were used. All M. bovis clinical isolates were previously characterized by spoligotyping (data not shown) and all M. tuberculosis by spoligotyping and MIRU (Noguti et al., 2010). Amplification was carried out with specific primers for M. tuberculosis complex TB1 (5′-CCTGCGAGCGTAGGCGTCGG-3′) and TB2 (5′-CTCGTCCAGCGCCGCTTCGG-3′) (Eisenach et al., 1990) which amplify a fragment of 123 base pairs (bp) of the IS6110 sequence and with specific primers for M. bovis pncATB-1.2 (5′-ATGCGGGCGT TGATCATCGTC-3′) and pncAMB-2 (5′-CGGTGTGC CGGAGAAGCCG-3′) (De Los Monteros et al., 1998; Bannalikar et al., 2006) which amplify a fragment of 186 bp of pncA gene. DNA extracts (1 μL) from all mycobacterial reference strains and clinical isolates, were amplified by multiplex-PCR using 24 μL reaction mixture containing 0.2 pmol/μL TB1 and TB2 primers (Invitrogen - Integrated DNA Technologies, Inc. Coralville, USA), 0.4 pmol/μL pncATB-1.2 and pncAMB-2 primers (Invitrogen - Integrated DNA Technologies, Inc. Coralville, USA) and PCR Master Mix (Promega Corporation, Madison, Wisconsin, USA), according to manufacturer’s instructions. Amplification was carried out in a Perkin-Elmer Gene Amp PCR System 2400 thermalcycler (Waltham, Massachusetts, USA) with an initial cycle of 5 min at 94 °C; 35 cycles of 1 min at 94 °C, 1 min at 68 °C, 1 min at 72 °C and a final extension at 72 °C for 10 min. M. tuberculosis H₃₇Rv (1 ng) and M. bovis (1 ng) as positive and water as negative controls were included in all assays. PCR products were separated by electrophoresis on 2% agarose gel (Biotools/M&B Laboratories, S. A., Uniscience do Brasil, São Paulo, Brazil) at 100 V for 1 h in TBE. Gel were stained with ethidium bromide 0.5 μg/μL, visualized under an ultraviolet light Macrovue (Pharmacia Bioscience, Upsala, Sweden), and photodocumented digitally by Power Shot S215 (Cannon, NY, USA). A 100 bp DNA standard (Invitrogen Life Technologies, São Paulo, Brazil) was used as a DNA size marker.

The multiplex-PCR showed to be specific to differentiate M. bovis from other MTC species by amplification of two DNA fragments sized 123 and 186 bp and a 123 bp DNA fragment respectively. No PCR product of specific sizes to M. tuberculosis and M. bovis were observed in amplification of DNA from nontuberculous mycobacteria reference strains except for M. avium that showed a DNA fragment sized more than 300 bp (Figure 1). Also, all M. tuberculosis and M. bovis clinical isolates (confirmed by spoligotyping) showed a single (123 bp) and two (123 and 186 pb) DNA fragments respectively, by multiplex-PCR.

Figure 1.

Specificity of multiplex-PCR for differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC). Electrophoresis on 2% agarose gel. Line M: DNA molecular weight marker (100 bp); Line 1: M. tuberculosis H37Rv; Line 2: M. bovis AN5; Line 3: M. bovis BCG; Line 4: M. avium; Line 5: M. kansasii; Line 6: M. abcessus; Line 7: M. chelonae; Line 8: M. massiliense; Line 9: M. szulgai; Line 10: M. fortuitum; Line 11: negative control.

The multiplex-PCR combining TB1/TB2 (Eisenach et al., 1990) and pncATB-1.2/pncAMB-2 (De Los Monteros et al., 1998) primers, proposed in this study, could not differentiate among the six species of MTC, but it showed to be specific in differentiating M. bovis from the others. Exception was for M. bovis BCG, which could not be differentiated from wild M. bovis (clinical isolates), once the first one tested has the 169C > G mutation too.

The correct differentiation of M. bovis triggers to an appropriate therapy, knowledge on TB epidemiology and control of bovine infection (De Los Monteros et al., 1998; Bannalikar et al., 2006; De La Rua-Domenech et al., 2006). In fact, the proposed multiplex-PCR seems to be a very useful tool for complementation in differentiating M. bovis from others MTC species, which are slow-growing mycobacteria, where a small number of bacilli in cultures hinder identification by biochemical tests. Additionally, biochemical identification methods are not able to provide satisfactory results and the multiplex-PCR also has the advantage of being a fast method with results provided in one day. As consequence, a specific, fast and low cost diagnosis method is highly relevant to differentiate M. bovis isolates from the M. tuberculosis complex (Bannalikar et al., 2006).

The multiplex-PCR showed to be an important tool for differentiation of M. bovis from M. tuberculosis cultured isolates tested. Hopefully, the proposed multiplex-PCR should be tested with the other species from the MTC to set up its potential in differentiating them as the multiplex-PCR proposed by Yeboah-Manu et al. (2001). The multiplex-PCR added of a internal control should also be studied in clinical specimens as additional diagnostic tool mainly in regions where bovine and human TB coexist for the monitoring infection by M. bovis in human beings.