Rapid and Specific Detection of Mycobacterium tuberculosis by Using the Smart Cycler Instrument and a Specific Fluorogenic Probe

Abstract

A procedure using the Smart Cycler instrument and a fluorescence quencher (FQ) probe for the specific identification of Mycobacterium tuberculosis complex (MTB) was used to detect organisms in 366 acid-fast bacillus smear-positive respiratory specimens. It was compared to culture and the AMPLICOR M. tuberculosis PCR test. MTB was isolated from 198 of these samples. The FQ PCR assay was sensitive (197 of 198, 99.5%) and specific (165 of 168, 98.2%); no significant difference was observed between the two PCR protocols. After DNA extraction, a final result was available within 1.5 h with the real-time PCR protocol.Because of the high risk of person-to-person transmission, rapid detection of Mycobacterium tuberculosis directly in patient specimens is recognized as an important step in the control of infection due to this organism (9, 12). The detection of acid-fast bacilli (AFB) in smears prepared from respiratory secretions has been the mainstay of rapid identification of potentially infectious individuals. Direct staining of prepared smears is generally performed with Ziehl-Neelsen stain or auramine fluorescent dye (8). Because the AFB smear lacks specificity, there is a need for a laboratory test for specific detection of the M. tuberculosis complex (MTB) that can be performed within a short period of time. Currently, nucleic acid amplification tests offer a rapid, specific, and sensitive approach to the detection and characterization of MTB (2,3,6,14,16,20,21). However, many of these assays require multiple user-dependent steps for amplification and detection and have the potential for error and sample contamination. Real-time PCR techniques (22), involving fluorescent dyes or fluorophores with a spectrofluorometric thermal cycler, have been developed and evaluated for the detection of mycobacteria and for the detection of drug resistance (5,11,18,19). The ABI 7700 TaqMan system (Applied Biosystems, Foster City, Calif.) has been used to quantitate MTB DNA in sputum during the treatment of tuberculosis patients (4). The probe used in this assay was specific for the IS6110 gene region (7).In addition, we recently reported a rapid and sensitive method for the identification of MTB by amplification of the internal transcribed spacer and the use of a specific fluorogenic probes for MTB in the LightCycler system (10, 15).In this study, we compared the fluorescence quencher (FQ) PCR assay to the AMPLICOR PCR test and conventional culture techniques. We examined AFB smear-positive respiratory specimens that were submitted for culture from January 2001 through December 2002. All specimens were liquefied and decontaminated with N-acetyl cysteine-2.5% NaOH and concentrated by centrifugation (17). The sediment was used to inoculate a selective 7H11 agar plate and a supplemented BacT/ALERT MP (MP; Organon Teknika, Durham, N.C.) culture bottle. Also, two smears were prepared and stained with an auramine fluorochrome dye (8). Isolates of mycobacteria growing on solid media were identified by DNA probes (Accuprobe; Gen-Probe, I...

Rapid detection of Mycobacterium tuberculosis complex by real-time PCR in sputum samples and its use in the routine diagnosis in a reference laboratory

“…Other studies also reported false-negative results by RT-PCR in paucibacillary samples (Miller et al, 2002;Cleary et al, 2003;Lira et al, 2013). These authors observed an inversely proportional relationship between the C t values and the number of AFB detected in microscopy, as also encountered in our study (data not shown).…”

“…The use of RT-PCR for TB diagnosis has increased, as it has proven to be highly sensitive and specific for detection of the Mycobacterium tuberculosis complex (MTBC) directly in clinical samples or from culture isolates (García-Quintanilla et al, 2002;Miller et al, 2002;Broccolo et al, 2003;Cleary et al, 2003;Armand et al, 2011;Lira et al, 2013). Many regions of the mycobacterial genome have been used in molecular diagnosis of TB, such as IS6110, 16S rRNA, hsp65, rpoB, sdaA, devR and mpt64 (Manjunath et al, 1991;Lachnik et al, 2002;Kim et al, 2004;Chakravorty et al, 2005;Negi et al, 2007;Hwang et al, 2013;Nimesh et al, 2013).…”

Evaluation of the Rapid MGIT TBc Identification Test for Culture Confirmation of Mycobacterium tuberculosis Complex Strain Detection

“…For the identification of M. tuberculosis complex strains in AFB-positive Bactec cultures (2 to 3 days after a positive signal was detected), IS6110 real-time PCR was performed according to the method of Cleary et al (9). The primers used for this assay were IS6 (5Ј-GGCTGTGGGTAGCAGAC C-3Ј) and IS7 (5Ј-CGGGTCCAGATGGCTTGC-3Ј), as well as an internal oligonucleotide probe (5Ј- [6-…”

Molecular-Beacon-Based Real-Time PCR for Detection and Quantification of Mycobacterium tuberculosis DNA in Clinical Samples

“…In the literature, TaqMan and linear fluorescence probes have mostly been applied for direct diagnosis of TB (1,3,5). MB technology has been evaluated for direct detection of M. avium (2, 6) as well as for detection of mutation-specific resistance in M. tuberculosis isolates (9, 10) and, in only one report, for direct TB diagnosis using pulmonary specimens (7).…”