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

  • Clinical Investigation
  • Published:
Japanese Journal of Ophthalmology Aims and scope Submit manuscript

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 ≥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.

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Acknowledgments

The authors thank Ikuyo Yamamoto, Shizu Inoue, and Chizuru Kato for providing technical assistance and the doctors of the Uveitis Group at Tokyo Medical and Dental University Hospital for sample collection. This work was supported by the Comprehensive Research on Disability, Health and Welfare, Health and Labor Sciences Research Grants, Ministry of Health, Labor and Welfare, Japan.

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Authors and Affiliations

  1. Department of Ophthalmology and Visual Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Manabu Ogawa, Sunao Sugita & Manabu Mochizuki

  2. Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan

    Sunao Sugita

  3. Division of Medical Science, Department of Virology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Norio Shimizu & Ken Watanabe

  4. Department of Section of Bacterial Pathogenesis, Graduate School of Medicine and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Ichiro Nakagawa

Authors
  1. Manabu Ogawa
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  2. Sunao Sugita
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  3. Norio Shimizu
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  4. Ken Watanabe
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  5. Ichiro Nakagawa
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  6. Manabu Mochizuki
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Corresponding author

Correspondence to Sunao Sugita.

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Ogawa, M., Sugita, S., Shimizu, N. et al. Broad-range real-time PCR assay for detection of bacterial DNA in ocular samples from infectious endophthalmitis. Jpn J Ophthalmol 56, 529–535 (2012). https://doi.org/10.1007/s10384-012-0174-z

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  • DOI: https://doi.org/10.1007/s10384-012-0174-z

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