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.