Affiliation:
1. Department of Medical Microbiology, University of Antwerp, Wilrijk, Belgium
2. bioMérieux, Boxtel, The Netherlands
Abstract
ABSTRACT
Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect
Mycoplasma pneumoniae, Chlamydophila pneumoniae
, and
Legionella
spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the
M. pneumoniae, C. pneumoniae
, and
Legionella pneumophila
16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of
M. pneumoniae
-,
C. pneumoniae
-, and
L. pneumophila
-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of
M. pneumoniae, C. pneumoniae
, and
Legionella
spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.
Publisher
American Society for Microbiology
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