Detection and Differentiation of
            <i>Mycobacterium tuberculosis</i>
            and Nontuberculous Mycobacterial Isolates by Real-Time PCR

Shrestha, Nabin K.; Tuohy, Marion J.; Hall, Gerri S.; Reischl, Udo; Gordon, Steven M.; Procop, Gary W.

doi:10.1128/jcm.41.11.5121-5126.2003

Cited by 106 publications

(84 citation statements)

References 29 publications

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“…The sensitivity and specificity of this assay for smear-positive respiratory specimens was 98.1% and 100% respectively with a turn-around time of less than 5 hours (Miller et al, 2002). Other studies, with different targets, have reported similar results (Shrestha et al, 2003) and many have been designed as multiplex assays to differentiate Mtb complex from other non-tuberculosis mycobacteria based on melting curve analysis (Shrestha et al, 2003). Furthermore, these assays were often shown to be as sensitive and specific as the available commercial assays such as the Cobas Amplicor (Miller et al, 2002;Shrestha et al, 2003).…”

Section: Laboratory-developed Pcr Methodssupporting

confidence: 70%

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

Babady¹,

Wengenack²

2012

Understanding Tuberculosis - Global Experiences and Innovative Approaches to the Diagnosis

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Section: Laboratory-developed Pcr Methodssupporting

confidence: 70%

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

Babady¹,

Wengenack²

2012

Understanding Tuberculosis - Global Experiences and Innovative Approaches to the Diagnosis

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“…Primers targeted to the 16S rRNA gene, RNA polymerase gene (rpoB), hsp65 gene or 16-23S internal transcribed spacer (ITS) region have been reported in relation to detecting mycobacteria by PCR in previous publications. As the HRMA assay could differentiate gene fragments of up to 300 bp, four pairs of genus-specific primers flanking gene fragments of different mycobacteria were selected from five previous studies (Foongladda et al, 2009;Kim et al, 2010;Richardson et al, 2009;Roth et al, 2000;Shrestha et al, 2003). The primer sequences are listed in Table S1 (available in JMM Online).…”

Section: Methodsmentioning

confidence: 99%

Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system

Perng

Chen

Chiueh

et al. 2012

Journal of Medical Microbiology

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Non-tuberculous mycobacteria (NTM) are increasingly important opportunistic pathogens responsible for a variety of clinical diseases. The aim of this study was to evaluate a novel technique, real-time PCR coupled with high-resolution melting analysis (real-time PCR-HRMA), for NTM identification. Two pairs of unique primers targeted to the 16S rRNA gene and the 16S-23S internal transcribed spacer region were selected for further evaluation. A total of 149 mycobacterial clinical isolates were subjected to analysis using the real-time PCR-HRMA system. Overall, 134 NTM identified by the 16S rRNA full-gene sequencing method were categorized into four major groups: Mycobacterium avium complex, Mycobacterium chelonae group, Mycobacterium gordonae and Mycobacterium fortuitum group. Of the 134 prevalent mycobacterial isolates, 101 mycobacteria (75.4 %) could be identified correctly by the real-time PCR-HRMA system. The individual sensitivities for the M. avium complex, M. chelonae group, M. gordonae and M. fortuitum groups were 90.9, 89.1, 100 and 36.8 %, respectively. The specificity of identifying these groups varied from 96.4 to 100 %. When identification failed, mostly it was attributable to various species in the M. fortuitum group. The real-time PCR-HRMA system is therefore a rapid and sensitive method for identifying prevalent NTM in a clinical laboratory.

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“…[20][21][22] Conventional PCR, and more recently real-time PCR, have been recognized as efficient tools for clinically applicable assays. 11,12,[23][24][25] The use of these tools is of particular interest in orthopedics, wherein the adhesion of bacteria within a biofilm on the surface of an implant may make the organism difficult to detect by conventional techniques. [15][16][17] Although some of these tools have been used to study orthopedic infections, much remains to be learned.…”

Section: Discussionmentioning

confidence: 99%

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections

Kobayashi

Bauer

Tuohy

et al. 2006

Journal Orthopaedic Research

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ABSTRACT:We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of ''molecular Gram stain'' with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results. ß

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Detection and Differentiation of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Isolates by Real-Time PCR

Cited by 106 publications

References 29 publications

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections

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