Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays.
ABSTRACT To develop and apply real-time PCR protocols to the detection and classification of the Gram status of bacterial pathogens in aqueous and vitreous humor collected from clinically suspected intraocular infections.
The analytical specificity of two PCR assays, SYBR Green 16S rDNA-Based Universal PCR (SGRU-PCR), and a Multiplex Gram-Specific TaqMan-Based PCR (MGST-PCR), was determined with 31 clinically important pathogens, including 20 Gram-positive and 11 Gram-negative. Analytical sensitivity was determined with a 10-fold dilution of Staphylococcus epidermidis and Escherichia coli DNA. Assays were further tested on aqueous (n = 10) and vitreous humor (n = 11) samples collected from patients with clinically diagnosed intraocular infections.
DNA was amplified from all control bacterial isolates when using SGRU-PCR. MGST-PCR correctly classified the Gram status of all these isolates. The SGRU-PCR limit of detection of S. epidermidis and E. coli DNA was 100 fg/μL (E = 0.82 and 0.86; r(2) = 0.99) and for MGST-PCR, 1 pg/μL (E = 0.66 and 0.70; r(2) = 0.99. For clinical intraocular samples, positivity of culture was 47.6% and for real-time PCR assays, 95.2%. Gram classification was achieved in 100% of MGST-PCR-positive samples. Among microbiologically negative samples, real-time PCR assays were positive in 90% of cases. The false-positive rate in control aqueous was 3.2%, and control samples of vitreous were negative.
The real-time PCR assays demonstrated good correlation, with culture-proven
With the use of these methods, bacterial detection was improved from 47.6% to 95.3%, demonstrating them to be sensitive, rapid tests for diagnosis of bacterial endophthalmitis.
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ABSTRACT: To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.PLoS ONE 10/2014; 9(10):e109061. · 3.53 Impact Factor
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ABSTRACT: To review the microbiology of culture-positive cases of bacterial endophthalmitis, and to correlate this with visual outcomes.06/2014;