DNA Extraction Methods for Panbacterial and Panfungal PCR Detection in Intraocular Fluids

Abstract

Purpose: Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification.

Material and Methods: Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out.

Results: The column-based extraction method was the most time-effective, achieving DNA detection limits ≥10² and 10³CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4).

Conclusions: The column-based extraction method for very small inocula in small volume samples (50–100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

• Coagulase-negative staphylococci
• DNA sequencing
• endophthalmitis
• intraocular infection
• molecular diagnosis
• PCR

Acknowledgements

The authors thank Mercè Miquel Jaureguibeitia (CRAG), Olga Riera and Carmen Torrecillas (Eye Bank for Blindness Treatment, Centro de Oftalmología Barraquer, Barcelona, Spain) for their important and expert help.

Declaration of interest

The authors declared no competing interests. This research was funded by Fondos de Investigaciones Sanitarias (FIS PI09/0992), Instituto de Salud Carlos III (Madrid, Spain).