Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay.
We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-mul glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.
Burggraf
Siegfried
S
Labor Becker, Olgemöller und Kollegen, Führichstrasse 70, D-81671 München, Germany. sburggraf@labor-bo.de
Reischl
Udo
U
Malik
Naeem
N
Bollwein
Markus
M
Naumann
Ludmila
L
Olgemöller
Bernhard
B
D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology*
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D011933:Reagent Kits, Diagnostic