Evaluation of real-time PCR assays, based in molecular beacon technology, for detection and quantification of Mycobacterium tuberculosis DNA
Abstract number: 1733_1482
Houhoula D.P., Siatelis A., Legakis N.J., Tsakris A., Papaparaskevas J.
Objective: The evaluation of a conventional and two real-time PCR assays, in comparison to the standard culture for tuberculosis (Tb), and the patients' clinical data.
Materials and Methods: A total of 60 clinical samples submitted for Tb diagnosis, were tested using three PCR assays targeting IS6110. The samples were 30 (7 pulmonary and 23 extra-pulmonary) from 22 patients, for which Tb diagnosis was based either on positive culture, or clinical presentation, and 30 from 30 patients with other non-Tb diagnoses. The assays were a previously reported (Kox et al, JCM 1994) conventional PCR (C-PCR), a real-time PCR (RT1) using previously reported nested-PCR inner primers (Caws et al, JCM 2000) with a newly designed molecular beacon, and a novel real-time PCR (RT2). The Beacon Designer 5.0 software was used for all newly designed beacons (RT1, RT2) and primers (RT2). DNA quantification was performed using a standard curve of five positive concentrations (3.4 pg/mL, and 340, 34, 3.4, and 0.34 fg/mL) of DNA extracted from M. tuberculosis H37Rv strain. All samples were tested neat and diluted 1/10 for PCR inhibition. Real-time PCR runs were acceptable when the negative controls had undetectable cycle threshold (CT) values, and the first two positive DNA concentrations had CT values of 27 to 30 cycles.
Results: Out of 30 samples from confirmed Tb cases, 28, 19 and 27 were positive using C-PCR, RT1, and RT2, respectively, corresponding to diagnostic sensitivities of 93%, 63% and 90%, respectively. Based on the DNA quantification performed, analytical sensitivities were 10 and 1 fg of DNA, for RT1 and RT2, respectively. When diluted specimens were examined, 5 inhibition cases were detected (1, 1 and 3 for C-PCR, RT1, and RT2, respectively). Combined performance of C-PCR+RT2 increased overall sensitivity to 100%. All 30 samples from non-Tb cases were negative with all assays (specificity 100%). RT2 quantification indicated that DNA extraction from tissue biopsies resulted in the lowest DNA yield compared to pleural/sputum samples (DNA load range 0.7659 fg, and 4.4576 pg, respectively). However, a single pus specimen with DNA load of 2.7 ng was identified.
Conclusions: RT2 and C-PCR were the most sensitive assays tested, and their combination resulted in 100% diagnostic sensitivity. Specificity was 100%. Real Time PCR, based on molecular beacon technology, seems to be a rapid and sensitive procedure for Tb diagnosis and DNA quantification in clinical samples.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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