ᰔAlthough real-time PCR (RT-PCR) has been extensively evaluated for diagnosis of Mycobacterium tuberculosis infections (5), data are limited on molecular-beacon (MB) applications. An MB-based RT-PCR protocol was designed and evaluated for direct M. tuberculosis detection and quantification in clinical specimens.A total of 1,019 samples (417 pulmonary and 602 extrapulmonary) were consecutively and prospectively collected for tuberculosis (TB) diagnosis. They were processed using standard methodology (4) and divided into two parts. The first half of each sample was used for acid-fast staining and culture, while the second half was stored at Ϫ70°C. TB diagnosis of patients followed previous definitions (11). After diagnosis elucidation, the second part of each specimen obtained from TB-positive patients, as well as an equal number of specimens obtained from TB-negative patients, was thawed and DNA was extracted using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany).The RT-PCR targeted the IS6110 sequence, and the following primers, amplifying a 161-bp fragment, and MB were designed, using Beacon Designer 5.1 software (Premier Biosoft, Palo Alto, CA): TB2F (5Ј-GTCCACGCCGCCAACTACG-3Ј), T〉2R (5Ј-GTTAGGTGCTGGTGGTCCGAAG-3Ј), and TB2B (6-carboxyfluorescein-5Ј-CGCGATCGCCACAG CCCGTCCCGCCGATGATCGCG-3Ј-benzoic acid succinimidyl ester). Conditions consisted of 2 min of denaturation at 95°C, 50 cycles of 45 s of denaturation at 93°C, 90 s of annealing at 60°C, and 2 min of extension at 72°C, and finally, 7 min of extension at 72°C. DNA extracted from the M. tuberculosis H37Rv strain (ATCC 25618) was used for the quantification standard curve. DNA from an M. avium and an M. chelonae clinical isolate, from an M. bovis BCG strain, and from Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, and Bacteroides fragilis ATCC 25285 strains was used for specificity
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