Nucleic acid sequence based amplification (NASBA) of Chlamydia pneumoniae major outer membrane protein (ompA) mRNA with bioluminescent detection.
Chlamydia pneumoniae has been associated with chronic conditions such as atherosclerosis and coronary heart disease but the precise role of this intracellular bacteria in the pathogenesis of these diseases is not well defined. Several techniques have been developed for detection of C. pneumoniae in atheromatous lesions, however it remains unclear whether persistent forms of the organism and/or actively replicating bacteria contribute to associated pathology. The aim of this study was to utilize nucleic acid sequence based amplification (NASBA) technology together with a highly sensitive aequorin bioluminescent hybridization assay for the detection of C. pneumoniae ompA mRNA transcripts. A NASBA targeting the ompA gene of C. pneumoniae was developed, and the sensitivity was evaluated using both C. pneumoniae ompA RNA generated in vitro, and purified C. pneumoniae inclusion forming units (IFU). C. pneumoniae NASBA was capable of detecting between 100 and 1000 ompA RNA molecules and could detect 0.2 IFU of C. pneumoniae using the aequorin bioluminescent assay. The sensitivity of the bioluminescent assay was at least 10-fold higher than Northern blot detection. The linearity of NASBA amplification was assessed in time-course amplification experiments with different input numbers of RNA molecules. When NASBA products were analyzed during the linear phase of amplification, the dynamic range of bioluminescent detection extended over 8-log units of input RNA copy number. NASBA amplification coupled with bioluminescent detection may prove to be a useful molecular tool for the detection, quantitation and analysis of differentially expressed chlamydial genes during various stages of infection and disease pathology or for other mRNAs of interest in different disease processes.
Coombes
B K
BK
The Father Sean O- Sullivan Research Center, St. Joseph-s Hospital, McMaster University, Hamilton, L8N 4A6, Canada. coombebk@fhs.mcmaster.ca
Mahony
J B
JB
D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001681:Biological Assay / Q000379:methods*; D016993:Chlamydophila pneumoniae / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008163:Luminescent Measurements; D021141:Nucleic Acid Amplification Techniques; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D001425:Bacterial Outer Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins; D000331:Aequorin