Japanese Journal of Ophthalmology

, Volume 56, Issue 6, pp 529–535 | Cite as

Broad-range real-time PCR assay for detection of bacterial DNA in ocular samples from infectious endophthalmitis

  • Manabu Ogawa
  • Sunao Sugita
  • Norio Shimizu
  • Ken Watanabe
  • Ichiro Nakagawa
  • Manabu Mochizuki
Clinical Investigation

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 Staphylococcusaureus and Escherichiacoli DNA.

Results

DNA extraction using the Bacteria card® without enzymes resulted in detection of all the tested strains at concentrations ≥107 copies/mL. Extraction with the Bacteria card® with lysozyme resulted in detection of all the tested strains at concentrations ≥106 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.

Keywords

Endophthalmitis Bacteria Polymerase chain reaction 

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Copyright information

© Japanese Ophthalmological Society 2012

Authors and Affiliations

  • Manabu Ogawa
    • 1
  • Sunao Sugita
    • 1
    • 2
  • Norio Shimizu
    • 3
  • Ken Watanabe
    • 3
  • Ichiro Nakagawa
    • 4
  • Manabu Mochizuki
    • 1
  1. 1.Department of Ophthalmology and Visual Science, Graduate School of Medical and Dental SciencesTokyo Medical and Dental UniversityTokyoJapan
  2. 2.Laboratory for Retinal RegenerationRIKEN Center for Developmental BiologyKobeJapan
  3. 3.Division of Medical Science, Department of Virology, Graduate School of Medical and Dental SciencesTokyo Medical and Dental UniversityTokyoJapan
  4. 4.Department of Section of Bacterial Pathogenesis, Graduate School of Medicine and Dental SciencesTokyo Medical and Dental UniversityTokyoJapan

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