Specificity of insertion sequence-based PCR assays for mycobacterium tuberculosis complex.
OBJECTIVE
To determine the specificity of different insertion sequence-targeted polymerase chain reaction (PCR) tests for Mycobacterium tuberculosis complex.


METHODS
One M. bovis BCG strain, two M. tuberculosis strains and ten species of mycobacteria other than tuberculosis (MOTT) were tested by three PCR assays based on the repetitive elements IS6110, IS1081 and IS990 under variable amplification conditions (different temperatures of primer annealing and numbers of reaction cycles).


RESULTS
DNA amplifications based on the three insertion sequences yielded fragments of expected sizes only in DNA from M. tuberculosis complex strains when the tests were conducted at high stringency (65 degrees C). At the annealing temperature of 60 degrees C the PCR assay with IS6110-specific primers yielded a 245 bp fragment also in nine MOTT strains tested. This could result from previously reported homology between non-tuberculous mycobacteria and a central region of IS6110. Amplification assays based on IS1081 and IS990 gave false-positive results in some MOTT isolates only under very low stringency (55 degrees C), which could be due to non-specific priming of the target DNA at that temperature.


CONCLUSIONS
Repetitive elements IS1081 and IS990 may represent a more reliable alternative to the more widely used IS6110 PCR target for tuberculosis diagnosis.
Dziadek
J
J
Centre for Microbiology and Virology, Polish Academy of Sciences, Lódź. jdziadek@wirus.cmiwpan.lodz.pl
Sajduda
A
A
Boruń
T M
TM
D004251:DNA Transposable Elements / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D004251:DNA Transposable Elements