Bacterial species identification after DNA amplification with a universal primer pair

The diagnosis of bacterial infections can be difficult and time consuming. Rapid and reliable molecular triage of potentially infected patients, parti cularly the young and the elderly, would prevent unnecessary hospitalizatio ns, reduce associated medical costs, and improve the quality of care. Polym erase chain reaction (PCR) amplification utilizing a universal bacterial pr imer pair, followed by hybridization with species-specific probes, would al low rapid identification of the presence or absence of bacterial DNA, along with an identification of the bacterial species present. Molecular microbi ological analyses will require access to bacterial strain standards that ca n be catalogued and distributed to clinical laboratories. We amplified temp late DNA in filter paper spots containing boiled bacteria from 14 clinical isolates using a universal primer pair for the 16S ribosomal RNA (rDNA) cod ing sequence. Species-specific probes were hybridized to the amplification products for bacterial species identification. We conclude that template DN A can be identified with species-specific probes after universal bacterial amplification with a single primer pair. We also demonstrate a rapid and ef ficient method for the long-term storage and cataloguing of bacterial DNA f or use in quality control at clinical laboratories adopting molecular diagn ostic methodologies. We speculate that PCR amplification combined with spec ies-specific probe hybridization not only will represent an improvement ove r culture-based methods in terms of speed, sensitivity, and cost, but will also allow for the identification of unculturable bacteria and emerging or reemerging pathogenic organisms. (C) 1999 Academic Press.