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.

The Antimicrobial Resistance and Prevalence of Enterococcus Species in Saudi Arabia

“…REP-PCR were established and demonstrated great outcomes when utilized to type Enterococcus strains 13 . There are a few other PCR-based strategies effectively applied for enterococcal separation, for example RAPD-PCR 28 , ITS-PCR 29 , tDNA-PCR 30 , AFLP 31 , and PCR-RFLP 32 . Additionally, multilocus arrangement examination (MLSA) is likewise a procedure utilized for recognizable proof of enterococci by utilizing partial sequences of RNA polymerase alpha subunit, phenylalanyl-tRNA synthase and the alpha subunit of ATP synthase 33,34 .…”

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis

“…A conventional PCR was also carried out with the universal primer set targeting 16S rRNA genes to compare the sensitivity of the LAMP assay. The paired primers were 5′-CCAGCAGCCGCGGTAATACG-3′ and 5′-ATCGG(C/ T)TACCTTGTTACGACTTC-3′ (Lu et al, 2000). Twentyfive microlitre of PCR assay contained 2 lL of DNA template, 1 lL of each primer, 2 mM MgCl 2 , 0.2 mM dNTPs, 2.5 lL of 10 9 buffer and 1.25 U Taq HS DNA polymerase (Takara Bio, Shiga, Japan).…”

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces

“…2001). In the assay, a pair of ‘universal’ primers is also included to detect a conserved sequence of 16S ribosomal DNA, which has been found to be conserved among at least 14 species of pathogenic bacteria (Lu et al. 2000).…”