title: Broad-range real-time PCR assay for detection of bacterial DNA in ocular samples from infectious endophthalmitis.
abstract: BACKGROUND
  To evaluate a broad-range real-time polymerase chain reaction (PCR) targeting the bacterial 16S rRNA gene for detection of bacterial DNA in infectious endophthalmitis.
  
  
  METHODS
  The bacterial 16S rRNA gene was measured by quantitative real-time PCR. For the assay, bacterial DNA was prepared from 12 Gram-positive and 4 Gram-negative strains. To determine the optimum method for DNA extraction, four extraction procedures were selected by using DNA extraction program cards with and without the use of lysozyme. To evaluate PCR sensitivity, PCR fragments were amplified from Staphylococcus aureus and Escherichia coli DNA.
  
  
  RESULTS
  DNA extraction using the Bacteria card(®) without enzymes resulted in detection of all the tested strains at concentrations ≥ 10(7) copies/mL. Extraction with the Bacteria card(®) with lysozyme resulted in detection of all the tested strains at concentrations ≥ 10(6) copies/mL, indicative of no significant difference between the two procedures. DNA extraction using the Virus card(®), both with and without enzymes, resulted in reduced efficiency of detection of all strains compared with use of the Bacteria card(®). The PCR could detect as few as 1-10 colony-forming units (CFU) in diluted vitreous samples per reaction, and all tested bacterial species known to cause endophthalmitis were detected.
  
  
  CONCLUSIONS
  Bacterial 16S-specific PCR can comprehensively detect the main causative bacteria of clinically suspected endophthalmitis.
authors.affiliation: Department of Ophthalmology and Visual Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Ogawa
authors.forename: Sunao
authors.initials: S
authors.lastname: Sugita
authors.forename: Norio
authors.initials: N
authors.lastname: Shimizu
authors.forename: Ken
authors.initials: K
authors.lastname: Watanabe
authors.forename: Ichiro
authors.initials: I
authors.lastname: Nakagawa
authors.forename: Manabu
authors.initials: M
authors.lastname: Mochizuki
mesh_terms: D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis*; D009877:Endophthalmitis / Q000382:microbiology*; D015818:Eye Infections, Bacterial / Q000382:microbiology*; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S