Detection of rRNA from four respiratory pathogens using an automated Qβ replicase assay

Ribosomal RNA targets from Mycobacterium avium complex (23S), Mycoplasma pneumoniae (16S), Pneumocystis carinii (18S) and Legionella pneumophila (16S) were detected in four separate assays on a model automated Q-beta amplification instrument. Sandwich hybridization, reversible target capture, detector probe amplification and fluorescent signal detection occurred in closed, disposable packs at 38°C. Packs were injected with 0.5 ml samples in 3.06 M guanidine thiocyanate. Ten samples per run were read after 7 h, requiring only 4 min loading time. Synthetic RNA transcripts and purified, natural RNAs from up to four different strains per assay were diluted to 10⁶ or fewer molecules per sample (approximately 100 cells for prokaryotes, 10 cells for Pneumocystis). All analytes were detected at 10⁶ targets. The limits of detection were found at 10⁵ to 10⁴. Discrimination against competitor RNA was tested using up to 10⁹ molecules (1000 X excess) of appropriate test strains. Samples containing either zero targets or 10⁷ competitors produced negative results in 95 to 100% of the samples, depending on the assay. Closely related Legionella and Mycoplasma species cross-reacted at high challenge levels of 10⁹ molecules as a result of sequence similarities in the target regions. These results demonstrate the utility and versatility of an automated, high sensitivity, closed system for amplified analysis of direct from sample testing of respiratory pathogens.