DETECTION OF RIBOSOMAL-RNA FROM 4 RESPIRATORY PATHOGENS USING AN AUTOMATED Q-BETA REPLICASE ASSAY

Ribosomal RNA targets from Mycobacterium avium complex (23S), Mycoplas ma pneumoniae (16S), Pneumocystis carinii (18S) and Legionella pneumop hila (16S) were detected in four separate assays on a model automated Q-beta amplification instrument. Sandwich hybridization, reversible ta rget capture, detector probe amplification and fluorescent signal dete ction occurred in closed, disposable packs at 38 degrees C. Packs were injected with 0.5 ml samples in 3.06 M guanidine thiocyanate. Ten sam ples per run were read after 7 h, requiring only 4 min loading time. S ynthetic RNA transcripts and purified, natural RNAs from up to four di fferent strains per assay were diluted to 10(6) or fewer molecules per sample (approximately 100 cells for prokaryotes, 10 cells for Pneumoc ystis). All analytes were detected at 10(6) targets. The limits of det ection were found at 10(5) to 10(4). Discrimination against competitor RNA was tested using up to 10(9) molecules (1000 X excess) of appropr iate test strains. Samples containing either zero targets or 10(7) com petitors produced negative results in 95 to 100% of the samples, depen ding on the assay. Closely related Legionella and Mycoplasma species c ross-reacted at high challenge levels of 10(9) molecules as a result o f sequence similarities in the target regions. These results demonstra te the utility and versatility of an automated, high sensitivity, clos ed system for amplified analysis of direct-from-sample testing of resp iratory pathogens. (C) 1996 Academic Press Limited