Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens.
METHODS
The rapid detection of Mycobacterium tuberculosis (TB) in clinical samples is an important goal. The LightCycler heralds an advance in thermal cycle technology combining rapid cycle DNA amplification with fluorimetry, eliminating the need to perform amplification and product analysis separately.


OBJECTIVE
To evaluate the LightCycler for direct detection of M. tuberculosis complex in respiratory specimens. To evaluate a DNA extraction method based on Chelex 100 resin, heating and ultrasonication for the prevention of endogenous inhibitions in respiratory samples.


METHODS
DNA was extracted from sputum samples using the Chelex method and polymerase chain reaction (PCR) for TB performed with the LightCycler.


RESULTS
For 88 sputum samples positive by microscopy and culture for M. tuberculosis, 95% were PCR-positive. None of the five sputum samples that were smear-negative but culture-positive for M. tuberculosis, the 79 culture-negative sputum samples and the 29 sputum samples that were culture-positive for mycobacteria other than TB yielded positive PCR results. PCR inhibitors were not detected in any of the samples.


CONCLUSIONS
The LightCycler proved a simple, reproducible and rapid system, reducing the time to result from weeks (culture) or days (conventional PCR) to hours. The Chelex 100 resin method produced good results for the smear-positive specimens. However, a larger study is required to determine the efficacy of the method with smear-negative specimens and for specimens known to contain endogenous inhibitors.
Heginbothom
M L
ML
Department of Medical Microbiology and Public Health Laboratory, University Hospital of Wales, Cardiff, United Kingdom. margaret.heginbothom@phls.wales.nhs.uk
Magee
J T
JT
Flanagan
P G
PG
D005470:Fluorometry; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013183:Sputum / Q000382:microbiology*