Development and evaluation of triplex PCR for direct detection of mycobacteria in respiratory specimens

Abstract

Aims: To develop a method for the simultaneous detection of Mycobacterium tuberculosis, Mycobacterium avium‐intracellularecomplex (MAC), and other mycobacteria in clinical specimens using triplex PCR. Methods and Results: The target of amplification was the internal transcribed spacer region between the l6S and 23S rDNA genes. Twenty‐two mycobacterial type strains, 118 M. tuberculosis, 87 other mycobacteria, 75 nonmycobacterial pathogens, 115 respiratory specimens from confirmed cases of tuberculous or other mycobacterial diseases, and sputa from 50 patients with nonmycobacterial respiratory diseases were tested. In M. tuberculosis, 322 bp pan‐mycobacterial and 133 bp species‐specific bands were observed. In MAC, the respective bands were 322 and 84 bp. The other mycobacteria showed single pan‐mycobacterial bands of approx. 300–350 bp. Nonspecific amplicons were not found in any of the nonmycobacterial pathogens. In the tuberculosis specimens, 96·4% of smear‐positive specimens and 70·2% of smear‐negative specimens showed positive reactions. Specimens from two patients with MAC infection were MAC positive. Only 1 of 50 specimens from nonmycobacterial diseases was positive (2·0%). Conclusion, Significance and Impact of the Study: Triplex PCR enables accurate and rapid diagnosis of tuberculosis and probably is useful for the detection of MAC and other mycobacteria in respiratory specimens.

Multiplex PCR assay for simultaneous detection and differentiation ofMycobacterium tuberculosis,Mycobacterium aviumcomplexes and other Mycobacterial species directly from clinical specimens

“…These technical concerns are competition and ⁄ or homology between the primers chosen, annealing temperature and optimum concentration of the primers used. Though different multiplex PCR assays have been reported for the differentiation of M. avium from M. intracellularae complex (Ruiz et al 2001), M. tuberculosis complex from M. bovis (Shah et al 2002;Pinsky and Banaei 2008) and M. tuberculosis complex from M. avium complex (Mustafa et al 1999;Tanaka et al 2003;Park et al 2006), ours was unique in that we could successfully differentiate M. tuberculosis and M. avium complexes from other mycobacterial species. Mustafa et al (1999) developed an m-PCR for the differentiation of MTB complex from other mycobacterial species with a sensitivity of 88%, whereas the present assay had sensitivity between 96AE0% and 100% depending on the nature of the clinical specimens (Table 3).…”

“…1999; Tanaka et al. 2003; Park et al. 2006), ours was unique in that we could successfully differentiate M. tuberculosis and M. avium complexes from other mycobacterial species Mustafa et al.…”

“…Mustafa et al (1999) developed an m-PCR for the differentiation of MTB complex from other mycobacterial species with a sensitivity of 88%, whereas the present assay had sensitivity between 96AE0% and 100% depending on the nature of the clinical specimens (Table 3). Even Park et al (2006) had reported m-PCR to differentiate M. tuberculosis from M. avium complexes with a sensitivity of 96AE4% and they used only respiratory samples, while our system had high sensitivity not only in the respiratory samples but also in various other clinical specimens including sputum, CSF, LNA, pleural fluid, blood and urine (Table 3). Our system not only worked well with all types of clinical samples but also equally useful in both-HIV positive (sensitivity of 98AE07%) and HIV-negative (97AE05%) cases.…”

Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

“…Previously reported PCR methods for genus detection seem to have different drawbacks apparently avoided by this new PCR. As alignment between published primers/probes and genomic targets used by other researchers (16S rRNA, ITS, and heat shock protein 65) suggested, some might not detect DNA from various mycobacteria (37,38), others would detect nonmycobacterial DNA (20,21,23,25,32), and others might combine both sensitivity and specificity limitations (22,24,26,39). For M. avium subspecies detection, we used the multicopy element IS1311, a target previously used in multiplex real-time PCR assays (25) that, as far as we know, is M. avium subspecies specific (5).…”

“…Multiplexing strategies allow for further improvement of diagnostics in terms of rapidity and efficiency, and multiplex real-time PCR represents a reliable alternative. Among the various multiplex PCR assays reported thus far (20)(21)(22)(23)(24)(25)(26), many are conventional PCRs or use melting curve analysis and need additional interpretation; some have not been used directly on clinical samples, and others seem to have some specificity or sensitivity issues. A recently published duplex real-time PCR assay for the detection of M. tuberculosis and M. avium complexes on human respiratory specimens has been shown to be reliable and cost-effective (27), but it could have an added value by including an additional target for genus detection.…”