PCR-Based Method To Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions

Abstract

, and "Mycobacterium tuberculosis subsp. canettii" ("M. canettii") (7, 29) (the name "M. canettii" is in quotation marks since it does not appear on the official List of Bacterial Names with Standing in Nomenclature [http://www.bacterio.cict.fr]). Members of the MtbC are highly related mycobacteria exhibiting remarkable nucleotide sequence level homogeneity despite varying in pathogenicity, geographic range, certain physiological features (such as colony morphology as well as profiles of resistance and susceptibility to inhibitors), epidemiology, and host preference (10,11,39). Notably, M. bovis has a wide host range but is primarily a bovid pathogen, goats are the natural host of M.caprae, and M. microti is most often isolated from small rodents, while M. tuberculosis is the predominant cause of human tuberculosis (2,3,39,44,45). However, each of the MtbC subspecies is known to infect humans (16,17,20,22,27,33,(43)(44)(45), and since most laboratories do not fully identify MtbC isolates, the true cause of tuberculosis in these patients and its source often remain undiscovered. An important health concern is the zoonotic transmission of some MtbC subspecies from animals to humans and vice versa. Of particular significance is the transmission of M. bovis to humans from cattle and unpasteurized milk as well as M. bovis BCG infection of immunocompromised individuals (1,21,27,33). M. bovis is naturally resistant to pyrazidamide, a first-line antituberculosis drug (31,36). Therefore, complete identification of MtbC isolates at the subspecies level is required in order to collect information on their epidemiology and also to enable appropriate patient treatment and public health measures.Various biological and molecular mycobacterial characteristics have been utilized to identify MtbC isolates but have limited applicability as MtbC taxonomical tools. Although certain Mycobacterium species-specific gene sequence differences work well to differentiate mycobacteria other than MtbC (MOTT) from each other and from the MtbC, to date none can discriminate the individual MtbC subspecies due to genetic invariance in the target loci (8,11,26,28,34,37,39,41). In contrast, a

Epidemiology of Mycobacterium Bovis Infection in Wild Boar (Sus Scrofa) From Portugal

“…For the first four genes the PCR protocol described by Huard et al (2003) was used. Five microliters of Taq buffer 103 (Fermentas, Burlington, Canada), 1.25 U Taq polymerase, 1 ml of each primer at 20 mM, and 1.25 mg of DNA were added to a solution containing 200 mM of each dNTP, 1.5 mM MgCl 2 , and 5% dimethyl sulfoxide (DMSO), in a final volume of 50 ml.…”

“…Polymerase chain reaction products were visualized by electrophoresis in 1% agarose gel with ethidium bromide and photographed under UV light (Alpha Imager, Alpha Innotech Corporation, San Leandro, California, USA). According to Huard et al (2003) and Bartos et al (2006), this set of genes allows for the identification of M. bovis, Mycobacterium caprae, other members of the Mycobacterium tuberculosis complex, members of the Mycobacterium avium complex, and other mycobacteria not belonging to these two complexes (Table 2).…”

Evaluation of a Single-Tube Multiplex Real-Time PCR for Differentiation of Members of the Mycobacterium tuberculosis Complex in Clinical Specimens

“…The existing methods are not always suitable; as conventional (biochemical) methods are highly subjective, high-pressure liquid chromatography (HPLC) can identify only M. bovis BCG (3), and DNA probe and amplification assays based on 16S rRNA gene sequences can identify specimens only as MTBC. Published molecular assays to differentiate members of the MTBC are not in realtime PCR formats (6,14,20), do not identify members other than M. tuberculosis, M. bovis, and M. bovis BCG (10, 15), and are not validated for use on clinical specimens (10,15,16). To date no single assay to perform this diagnostic test exists for probe-based differentiation of members of the MTBC directly from clinical specimens.…”

“…The MTBC-RD real-time PCR targets RD (RD1, RD4, RD9, and RD12) (2,4,6,18) with primers and probes designed using Primer Express v. 3.0 (Applied Biosystems, Inc.…”

“…Differentiation between the species within this complex is important for public health surveillance and reference testing, as most species within the MTBC have been reported to infect humans (6). Additionally, it may be important information for physicians to rapidly identify severe side effects of M. bovis BCG in patients with bladder cancer or in vaccinated individuals or to assess transmission of M. bovis from animals or animal products.…”