Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains

Abstract

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC).Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.Tuberculosis (TB) is the leading cause of death worldwide from an infectious agent (13), with the WHO estimating that one-third of the global population is infected with Mycobacterium tuberculosis. In a global report from the WHO (2009), it was estimated that there were 9.27 million cases of TB in 2007, with 2 million associated deaths (41a). TB in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). The eight closely related species of the MTC have a wide range of natural hosts, including human hosts (M. tuberculosis, M. africanum, M. canettii), bovine hosts (M. bovis), caprine hosts (M. caprae), rodent hosts (M. microti), and pinniped hosts (M. pinnipedii), along with the attenuated M. bovis strain BCG (Bacillus Calmette-Guérin), the commonly used vaccine strain. While there are a number of natural hosts, each species of the MTC has been implicated in human infection (6,20).Traditionally, diagnosis of TB relies on smear microscopy and culture techniques in combination with a battery of biochemical tests which are time-consuming and labor-intensive and which often yield unreliable results (18). Nucleic acid diagnostic (NAD) techniques, in particular, real-time PCR, offer rapid, reliable, and highly sensitive alternative tools for the detection of many infectious agents (25, 42). Advances in real-time PCR, such as the availability of multiple fluorophores, along with the development of nonfluorescent quenchers, have facilitated multiplexing, allowing the simultaneous detection and differentiat...

Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections

“…There is currently no single nucleic acid diagnostics target described in the literature that can unambiguously identify H. influenzae. As such, in this study, a number of gene targets, including ssrA and lepA and genes that have previously been described in the literature as suitable bacterial species specific molecular diagnostics targets, were evaluated in silico (26)(27)(28). A putative novel diagnostics gene target, smpB, selected and identified by the NADRL, was also evaluated in silico.…”

“…These gene targets were chosen, as they are either present in all bacteria sequenced to date and/or have previously been demonstrated to be suitable as bacterial species-specific molecular-based diagnostics targets (26,28,36,37). Of these, only the smpB gene demonstrated sufficient in silico nucleotide sequence heterogeneity to allow for the design of an H. influenzae-specific diagnostic assay.…”

Seek TB, a Two-Stage Multiplex Real-Time-PCR-Based Method for Differentiation of the Mycobacterium tuberculosis Complex

“…The novel diagnostic targets used in this study were identified by using in silico comparative genomic approaches previously described (22,23). All oligonucleotides used in this study were designed in accordance with previously reported guidelines (10,24).…”

“…The panel of MTC isolates, nontuberculosis mycobacteria (NTM), and other bacterial species used in this study (see Tables S1 and S2 in the supplemental material) was the same panel as that described in a previous study (23), with the addition of 47 MTC isolates provided by the National Reference Center for Mycobacteria, Borstel, Germany (characterized as described previously by Allix-Bé-guec et al [2]). All genomic DNA samples used in this study were isolated, quantified, and stored as described previously (22).…”

“…Previously, we have reported the design and development of two 4-plex real-time PCR diagnostic assays for the accurate iden-tification of the MTC, one for the identification of M. tuberculosis and M. canettii and one for the identification of M. bovis, M. bovis BCG, and M. caprae (22,23). In this study, additional diagnostic targets were incorporated into these 4-plex assays, resulting in two 5-plex real-time PCR diagnostic assay.…”