Real-time PCR with internal amplification control for detecting tuberculosis: method design and validation.
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.
Flores
E
E
Section of Microbiology, Hospital General Universitario, Universidad Miguel Hernández, Elche, Spain.
Rodríguez
J C
JC
Garcia-Pachón
E
E
Soto
J L
JL
Ruiz
M
M
Escribano
I
I
Royo
G
G
D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013183:Sputum / Q000737:chemistry / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology / Q000652:urine
D004269:DNA, Bacterial