Burden of Mycobacterium tuberculosis in sputum samples can be reliably determined using a quantitative, non-radioactive polymerase chain reaction assay.
OBJECTIVE
To develop a rapid assay for quantitation of Mycobacterium tuberculosis in sputum samples using the competitive polymerase chain reaction (PCR) and a colorimetric microtiter well detection format.


METHODS
The assay relies on the co-amplification of a 419 base pair (bp) pab fragment of M. tuberculosis together with a target template (pab/tet) made by splicing a fragment of tet excised from pbr322 between the 5' and 3' ends of the pab fragment to create a 380 bp hybrid template amplified with the same primers but readily distinguishable using probes specific for either pab or tet.


RESULTS
We demonstrate a good correlation between the results obtained using this assay and the results of quantitative culture.


CONCLUSIONS
This assay provides quantitative information regarding M. tuberculosis burden in samples containing between 10(3) and 10(8) colony forming units/milliliter (CFU/ml).
Sweet-Cordero
E A
EA
Division of Infectious Diseases, University of California, San Francisco, USA.
Chambers
H F
HF
Cicero-Sabido
R
R
Santos-Preciado
J I
JI
D000818:Animals; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D013183:Sputum / Q000382:microbiology*