Detection of rRNA from four respiratory pathogens using an automated Q beta 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 degrees 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 10(6) or fewer molecules per sample (approximately 100 cells for prokaryotes, 10 cells for Pneumocystis). All analytes were detected at 10(6) targets. The limits of detection were found at 10(5) to 10(4). Discrimination against competitor RNA was tested using up to 10(9) molecules (1000 X excess) of appropriate test strains. Samples containing either zero targets or 10(7) 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 10(9) 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.

Comparison of Mycobacterium 23S rRNA sequences by high-temperature reverse transcription and PCR.

We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium. We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus. A 70 degrees C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with alpha-33P-labeled primers, again with extension at 70 degrees C. Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase. Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and Rhodococcus bronchialis as the outgroup. Our 23S rRNA trees were compared with previously published 16S rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously. Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed.