PCR based detection of Mycobacterium tuberculosis: effect of sample preparation.
Southeast Asian J Trop Med Public Health
;
1994 Dec; 25(4): 693-7
Article
in English
| IMSEAR
| ID: sea-31711
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.
Full text:
Available
Index:
IMSEAR (South-East Asia)
Main subject:
Sputum
/
Sri Lanka
/
Tuberculosis, Pulmonary
/
Humans
/
DNA, Bacterial
/
Gene Amplification
/
Polymerase Chain Reaction
/
Predictive Value of Tests
/
Mycobacterium tuberculosis
Type of study:
Diagnostic study
/
Practice guideline
/
Prognostic study
Country/Region as subject:
Asia
Language:
English
Journal:
Southeast Asian J Trop Med Public Health
Year:
1994
Type:
Article
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