Real-time PCR for broad detection of the Mycobacterium tuberculosis complex and medically important atypical mycobacteria
Abstract number: O239
Abdeldaim G., Carl-Johan R., Svensson E., Kirsebom L., Blomberg J., Herrmann B.
Objectives: To determine the sequence of the rnpB gene, coding for the ubiquitously present ribonuclease P RNA, and develop a real-time PCR for detection of the Mycobacterium tuberculosis complex and atypical mycobacteria.
Methods: The rnpB sequences of 17 Mycobacterium spp. were determined. Based on obtained rnpB sequences, two quantitative real-time PCRs for detection of the M. tuberculosis complex (Mytu PCR) and atypical mycobacteria (Myat PCR) were developed and combined into a single tube format. The analytical sensitivity of the PCR assay was determined with serial dilutions of target DNA. The specificity of the duplex PCR assay was tested with 21 mycobacteria species (55 strains), and 35 bacteria species other than mycobacteria. The PCR assay was evaluated on 10 samples from a quality control panel (QCMD) and on 442 clinical samples. The results were compared with the results of culture, direct microscopic examination and the Roche Amplicor PCR.
Results: Obtained rnpB sequences showed hypervariable regions enabling species specific identification and PCR design. The analytical sensitivity for detection of the M. tuberculosis complex was <50 copies/reaction, while for atypical mycobacteriae it was 500 copies/reaction. The assay was specific and did not detect any of 35 non-mycobacterial bacteria spp. The Mytu PCR specifically detected all four species of the M. tuberculosis complex and the Myat PCR detected all tested 17 atypical mycobacteria species, except 2 of 7 strains from the M. avium complex.
The PCR assay correctly detected all 10 samples from QCMD quality assurance panel. In analysis of 442 clinical specimens M. tuberculosis was detected in 40 cases (9%) by Mytu PCR, in 38 cases (8%) by the Roche PCR, in 46 cases (10%) by culture and in 23 (5%) cases by direct microscopic examination. The atypical mycobacteriae were detected by Myat PCR in 18 (4%) cases, in 11 (2%) by culture cases, and in 8 cases (2%) by direct microscopic examination.
Conclusions: Sequence determination of the rnpB gene is useful for Mycobacterium species identification.
Our duplex real-time PCR was shown to detect strains from both the M. tuberculosis complex and tested atypical mycobacteria strains. The PCR assay has a broader detection range of mycobacteria than commercial standard PCR method and has a sensitivity similar to culture.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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