Quantitation of Chlamydia trachomatis 16S rRNA using NASBA amplification and a bioluminescent microtiter plate assay

We developed a nucleic acid sequence based amplification (NASBA) assay whic h employs the recombinant photoprotein Aequorin in a microtiter plate forma t for detection and quantitation of C. trachomatis that may be useful in la rge scale epidemiological studies aimed at improving our understanding of f actors affecting transmission of this sexually transmitted pathogen. The co nditions for NASBA amplification of the 16S rRNA target were optimized (90 mM KCl, 12 mM MgCl2, 0.2 mu M P1 and P2 primers), amplified RNA was capture d by a biotin-labelled capture probe immobilized onto streptavidin coated m icrotiter plates and detected with an FITC-labelled oligonucleotide probe a nd Aequorin-anti-FITC antibody conjugate. The analytical sensitivity of NAS BA was 1,000 in vitro generated RNA transcripts and 1.6 IFU of C. trachomat is. The sensitivity of NASBA using the bioluminescent assay was 10 fold hig her than Northern blotting. Time course amplification experiments performed with 10 fold serial dilutions of target established that amplification was linear at 75 min and extended over a range of five log units of input RNA copy number. Linear regression analysis confirmed a linear fit for the data with r(2) = 0.959 (p < 0.004). A double log plot of RLU signal versus copy number was linear; analysis of residuals from a series of runs tests confi rmed a fit with a linear model (number of runs = 3, p = 0.5 where p < 0.05 indicates statistical deviation from a linear model). NASBA amplification c oupled with bioluminescent detection in a microtiter plate format should pr ovide a useful tool for quantitation of C. trachomatis in clinical specimen s for use in epidemiological studies.