Real-time PCR with internal amplification control for detecting tuberculosis: method design and validation

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Abstract:

Flores E, Rodríguez JC, Garcia-Pachón E, Soto JL, Ruiz M, Escribano I, Royo G. Real-time PCR with internal amplification control for detecting tuberculosis: method design and validation. APMIS 2009; 117: 592–7.

Real-time PCR has been a major development in the diagnosis of tuberculosis. However, most tests do not include an internal amplification control (IAC), which therefore limits it clinical application. In this study a new, easy to perform real-time PCR test with IAC was designed and validated in clinical samples. The primers amplified a 163-bp fragment of IS6110 of Mycobacterium tuberculosis and the IAC was designed with a fragment of a different microorganism (Chlamydia trachomatis). The interassay and intraassay variation of this test were very low (0.45–1.65% and 0.18–1.80%, respectively). The detection accuracy was validated in 50 samples (25 urine, 25 sputum) with different concentrations of M. tuberculosis, 18 clinical isolates of non-tuberculous mycobacteria and 148 samples with clinical suspicion of pulmonary tuberculosis. The specificity was 100%. The detection limit of this PCR test without IAC was approximately 15 bacteria and with IAC approximately 32 bacteria. This real-time PCR with IAC assay can improve the detection of M. tuberculosis and contribute to standardization of this diagnostic technique.

Keywords: Mycobacterium tuberculosis; diagnostic; internal control; real-time PCR

Document Type: Research Article

DOI: http://dx.doi.org/10.1111/j.1600-0463.2009.02508.x

Affiliations: 1: Section of Microbiology 2: Section of Pneumology 3: Research Unit, Hospital General Universitario, Universidad Miguel Hernández, Elche, Spain

Publication date: August 1, 2009

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