Detection of Mycobacterium tuberculosis by a polymerase chain reaction colorimetric dot-blot assay.
METHODS
A public health laboratory in a tuberculosis-endemic region in Brazil.


OBJECTIVE
To evaluate the accuracy of a combined polymerase chain reaction (PCR) colorimetric dot-blot protocol for Mycobacterium tuberculosis detection in clinical samples in a public health laboratory.


METHODS
Eighty clinical samples (13 cerebrospinal fluid, 31 induced sputum, 17 expectorated sputum, eight bronchoalveolar lavage and 11 pleural fluid) were assayed with the developed protocol. The accuracy of polymerase chain reaction (PCR) dot-blot methodology was compared to PCR agarose gel electrophoresis (PCR-AG) using as a gold standard the bacteriological result (culture and biochemical identification) combined with clinical follow-up. One internal region of the IS6110 repetitive element of the M. tuberculosis complex was selected for amplification and the amplified product transferred to nylon membranes to be detected by biotinylated DNA probe.


RESULTS
Overall sensitivity and specificity obtained were respectively 90% and 97% for PCR-AG and 95% and 97% for the PCR dot-blot. Among the 56 respiratory specimens, the sensitivity and specificity results for PCR-AG were respectively 88% and 95%, and for PCR dot-blot they were 94% and 95%. Among the 24 non-respiratory specimens the sensitivity and specificity results were respectively 83% and 100% for PCR-AG, and 100% and 100% for the PCR dot-blot protocol.


CONCLUSIONS
The results demonstrated that the PCR dot-blot assay may be helpful in the diagnosis of tuberculosis, and feasible even in resource-poor countries.
Sperhacke
R D
RD
Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde-Porto Alegre, RS, Brazil.
Mello
F C Q
FC
Zaha
A
A
Kritski
A
A
Rossetti
M L R
ML
D015139:Blotting, Southern / Q000379:methods*; D003124:Colorimetry / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology