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 epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Mycobacterium tuberculosis, Legionella pneumophila, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Haemophilus influenzae, and Neisseria meningitidis. The sizes of the amplified products from various bacteria were the same (996 bp), but the restriction patterns of most PCR products generated by HaeIII digestion were different. PCR products from S. aureus and S. epidermidis could not be digested by HaeIII but yielded different patterns when they were digested with MnlI. PCR products from S. pneumoniae, E. faecium, and E. faecalis yielded the same HaeIII digestion pattern but could be differentiated by AluI digestion. PCR products from E. coli, K. pneumoniae, S. marcescens, and E. cloacae also had the same HaeIII digestion pattern but had different patterns when digested with DdeI or BstBI. This universal PCR could detect as few as 10 E. coli or 250 S. aureus organisms. Compared with culture, the sensitivity of this universal PCR for detection and identification of bacteria directly from 150 cerebrospinal fluids was 92.3%. These results suggest that this universal PCR coupled with restriction enzyme analysis can be used to detect and identify bacterial pathogens in clinical specimens.
Lu
J J
JJ
Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, Republic of China. JJL@NDMCTSGH.EDU.TW
Perng
C L
CL
Lee
S Y
SY
Wan
C C
CC
D000368:Aged; D000369:Aged, 80 and over; D015373:Bacterial Typing Techniques; D002675:Child, Preschool; D017931:DNA Primers; D004275:DNA, Ribosomal; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D016920:Meningitis, Bacterial / Q000134:cerebrospinal fluid*; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S
D017931:DNA Primers; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S