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

Abstract

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.

FIG. 1

HaeIII digestion patterns of universal PCR products. Samples in different lanes were HaeIII-digested PCR products from the following bacteria: lane 1, S. aureus; lane 2, S. epidermidis; lane 3, S. pyogenes; lane 4, S. agalactiae; lane 5, S. pneumoniae; lane 6, E. faecium; lane 7, E. faecalis; lane 8, M. tuberculosis; lane 9, L. pneumophila; lane 10, E. coli; lane 11, K. pneumoniae; lane 12, S. marcescens; lane 13, E. cloacae; lane 14, P. aeruginosa; lane 15, A. baumannii; lane 16, P. mirabilis; lane 17, H. influenzae; lane 18, N. meningitidis. Lane M contained molecular size standards (base pairs). The sizes of the molecular size standards are marked on the left of the gel.

FIG. 2

Restriction digestion patterns of the universal PCR products. (A) MnlI digestion patterns of the universal PCR products from S. aureus (lane 1) and S. epidermidis (lane 2). (B) AluI digestion patterns of the universal PCR products from S. pneumoniae (lane 1), E. faecium (lane 2), and E. faecalis (lane 3). (C) DdeI digestion patterns of the universal PCR products from E. coli (lane 1), K. pneumoniae (lane 2), S. marcescens (lane 3), and E. cloacae (lane 4). (D) BstBI digestion patterns of the universal PCR products from E. coli (lane 1), K. pneumoniae (lane 2), S. marcescens (lane 3), and E. cloacae (lane 4). Lanes M are the same as in Fig. 1.

FIG. 3

Determination of the sensitivity of the universal PCR. Serial 10-fold dilutions of E. coli and S. aureus samples were amplified with the universal PCR, and the PCR products were electrophoresed on an agarose gel. (A) PCR results with E. coli DNA from 10⁸ to <10 organisms (lanes 1 to 9) and no bacteria (lane 10). (B) PCR results with S. aureus from 2.5 × 10⁷ to <10 organisms (lanes 1 to 9) and no bacteria (lane 10). Molecular marker sizes (lanes M) are given in base pairs on the left side of the picture. A band of approximately 150 bp is also seen in lanes that have the 996-bp PCR product; this band may be the result of nonspecific amplifications. Another band of approximately 50 bp is also present in some lanes with the intensity inversely proportional to the number of bacteria used for the PCR; this band is the primer dimer that formed during the PCR.

FIG. 4

Flow chart of the universal PCR and RFLP for detection and identification of common bacterial pathogens in body fluids.