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DNA extraction methods for panbacterial and panfungal PCR detection in intraocular fluids
Paloma Mazoteras1, Paulo José Martins Bispo, Ana Luisa Höfling-Lima
1Department of Surgery, School of Medicine, University of Barcelona (UB) , Barcelona , Spain .
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 ≥10(2) and 10(3 )CFU/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.
Insights
The column-based DNA extraction method is efficient for detecting bacterial and fungal DNA in intraocular fluids using PCR. This method improves pathogen detection rates in suspected endophthalmitis cases compared to traditional techniques.
Area of Science:
- Ophthalmology
- Molecular Biology
- Infectious Diseases
Background:
- Endophthalmitis diagnosis relies on identifying causative microorganisms.
- Traditional microbiological methods have limitations in sensitivity and speed.
- Molecular techniques like PCR offer potential for rapid and sensitive pathogen detection.
Purpose of the Study:
- To compare the efficiency of three DNA extraction methods from intraocular fluids.
- To evaluate the subsequent detection of bacterial and fungal DNA using universal PCR amplification.
- To determine the optimal method for diagnosing endophthalmitis.
Main Methods:
- Three DNA extraction methods were tested on aqueous and vitreous humors.
- Bacterial (E. coli, S. epidermidis) and fungal (C. albicans) strains were used for validation.
- DNA quality, quantification, and PCR detection limits were analyzed.
Main Results:
- The column-based method was most time-effective, with detection limits of ≥10^2 CFU/100µL for bacteria and 10^3 CFU/100µL for fungi.
- PCR detected 90.0% of causative agents, outperforming standard microbiological techniques (60.0%).
- Streptococcus spp. and Staphylococcus spp. were the most frequently identified organisms.
Conclusions:
- The column-based DNA extraction method is suitable for small-volume intraocular samples.
- This method enables PCR amplification with sufficient quality for microorganism identification.
- It offers improved diagnostic yield for suspected endophthalmitis.
