title: PCR based detection of Mycobacterium tuberculosis: effect of sample preparation.
abstract: Tests based on the polymerase chain reaction (PCR) for the detection of the Mycobacterium tuberculosis complex in clinical samples have a lower sensitivity when compared to culture. This has been attributed to the presence of inhibitors to Taq polymerase and/or suboptimal DNA extraction procedures. We tested different methods of processing smear negative culture positive sputum (n = 52) using different detergents, including nonidet P-40 (NP-40), sodium dodecyl sulphate (SDS), tween 20, triton X 100 and N-lauryl sarcosine. The detergents were used in combination with lysozyme and proteinase K enzymes. NP-40 was significantly better than SDS, tween 20 and N lauryl sarcosine (p < 0.05). When NP-40 was used as the detergent, 42 out of 52 specimens gave positive results with the standard amplification protocol which amplifies a 245 bp sequence of the insertion element IS 986. The 10 specimens that were negative were further diluted ten fold and/or eluted in sephadex G-50 columns before standard DNA amplification. A further 8 specimens then became positive. Elution in sephadex G-50 was better than ten fold dilution in processing of samples. The two negative samples had very low colony counts (n < 5). The study demonstrates that the sensitivity of the PCR is dependent on the sample preparation technique and the amount of target sequence available for amplification.
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Colombo, Sri Lanka.
authors.forename: J
authors.initials: J
authors.lastname: Perera
authors.forename: N V
authors.initials: NV
authors.lastname: Chandrasekharan
authors.forename: E H
authors.initials: EH
authors.lastname: Karunanayake
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics; D005784:Gene Amplification; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D013183:Sputum / Q000382:microbiology; D013188:Sri Lanka; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial