Journal Article

Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR

Humphrey, Bruce, Dark, Paul M., Warhurst, Geoffrey, Sutton, J. Mark, McLeod, Neil, Turner, Carrie

2015

Overview

More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated. We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Antimicrobial agents / Assaying / Azides - chemistry / Bacteria / Blood / Contaminants / Contamination / Copy number / Decontamination / Decontamination - methods / Deoxyribonucleic acid / Diagnostic systems / DNA / DNA Contamination / DNA Primers - chemistry / DNA Primers - genetics / DNA, Bacterial - chemistry / DNA, Bacterial - genetics / E coli / Gene Dosage - genetics / Genomes / Humans / Indicators and Reagents - chemistry / Inflammation / Medical research / Microorganisms / Molecular Sequence Data / Oligonucleotides / Pollutant removal / Polymerase chain reaction / Primers / Propidium - analogs & derivatives / Propidium - chemistry / Public health / Reagents / Real-Time Polymerase Chain Reaction - methods / Ribosomal DNA / Sensitivity / Sensitivity and Specificity / Sepsis / Strategy / Streptococcus infections / Studies / Systematic review / Ultraviolet radiation / Ultraviolet Rays