PCR PRIMERS AND PROBES FOR THE 16S RIBOSOMAL-RNA GENE OF MOST SPECIESOF PATHOGENIC BACTERIA, INCLUDING BACTERIA FOUND IN CEREBROSPINAL-FLUID

A set of broad-range PCR primers for the 16S rRNA gene in bacteria wer e tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and cons ists of a universal bacterial probe, a gram-positive probe, a Bacteroi des-Flavobacterium probe, and two probes for other gram-negative speci es. The second series was designed to detect PCR products from seven m ajor bacterial species or groups frequently causing meningitis: Neisse ria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria mon ocytogenes, and Staphylococcus aureus. The third series was designed f or the detection of DNA from species or genera commonly considered pot ential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-ne gative Staphylococcus spp. The primers amplified DNA from all 124 diff erent species of bacteria tested. Southern hybridization testing of th e broad-range probes with washes containing 3 M tetramethylammonium ch loride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococ cus radiopugnans and Gardnerella vaginalis. The gram-negative and gram -positive probes hybridized to isolates of two newly characterized bac teria, Alloiococcus otitis and Rochalimaea henselii, as predicted by G ram stain characteristics. The CSF pathogen and contaminant probe sequ ences were compared with available sequence information and with seque ncing data for 32 different species. Testing of the CSF pathogen and c ontaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus pro bes, these probes provided the correct identification of bacterial spe cies known to be found in CSF.