Use of PCR with universal primers and restriction endonuclease digestions for detection and identification of common bacterial pathogens in cerebrospinal fluid

We have designed a universal PCR capable of amplifying a portion of the 16S rRNA gene of eubacteria, including Staphylococcus aureus, Staphylococcus e pidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Mycobacterium tub erculosis, Legionella pneumophila, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Enterobacter cloacae, Pseudomonas aeruginosa, Acineto bacter baumannii, Proteus mirabilis, Haemophilus influenzae, and Neisseria meningitidis, The sizes of the amplified products from various bacteria wer e the same (996 bp), but the restriction patterns of most PCR products gene rated by HaeIII digestion were different, PCR products from S. aureus and S . epidermidis could not be digested by HaeIII but yielded different pattern s when they were digested with MnlI, PCR products from S. pneumoniae, E. fa ecium, and E. faecalis yielded the same HaeIII digestion pattern but could be differentiated by AluI digestion. PCR products from E. coli, K. pneumoni ae, S. marcescens, and E. cloacae also had the same HaeIII digestion patter n but had different patterns when digested with DdeI or BstBI, This univers al PCR could detect as few as 10 E. coli or 250 S. aureus organisms. Compar ed with culture, the sensitivity of this universal PCR for detection and id entification of bacteria directly from 150 cerebrospinal fluids was 92.3%. These results suggest that this universal PCR coupled with restriction enzy me analysis can be used to detect and identify bacterial pathogens in clini cal specimens.