Development of a 23S rRNA-based PCR assay for the detection of mycobacteria.
The partial nucleotide sequence of a recombinant plasmid containing the 23S rRNA gene of Mycobacterium tuberculosis was determined and an assay was developed for amplifying 23S rRNA gene sequences of mycobacteria. The PCR-based non-radioactive test enabled us to distinguish Mycobacterium from other closely related genera and was sensitive enough to detect 2 bacterial genome equivalents. The assay was extended to the detection of mycobacterial DNA in uncultured clinical specimens; 23S rRNA sequences were detected in thirty four of forty eight (70.8%) sputum and cerebrospinal fluid (CSF) specimens by the PCR assay, whereas direct smear examination and culture methods demonstrated a positivity rate of 29.2% and 16.7% respectively for the same specimens. A RNA-based PCR assay with a detection limit of 1 genome equivalent was also developed. These PCR assays should prove useful for the early and rapid detection of mycobacterial infection in uncultured clinical specimens.
Verma
A
A
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi.
Rattan
A
A
Tyagi
J S
JS
D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D012680:Sensitivity and Specificity
D012338:RNA, Ribosomal, 23S