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

Bruce Humphrey,Carrie Turner,J Mark Sutton,Geoffrey Warhurst,Neil Mcleod,Paul M Dark,Stefan J Green

doi:10.1371/journal.pone.0132954

Bruce Humphrey, Carrie Turner + Show 5 more

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https://doi.org/10.1371/journal.pone.0132954

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Abstract

BackgroundMore 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.ResultsWe 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.

Highlights

Molecular diagnostics aimed at the rapid detection of infectious diseases have become a powerful tool in modern medicine, with over 160 products currently approved by the FDA
We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents
Effect of amplicon length on qPCR reagent decontamination with ethidium monoazide Efficiency of contaminant DNA inactivation by treatment with increasing concentrations of EMA was investigated for bacterial 16S ribosomal RNA gene amplicons of different sizes using two primer sets (16SF-R = 169bp [19] and 9F-1116R = 1108bp [20]) from two papers conducting similar work, and a newly designed pan-bacterial primer pair (SF3c-SR5) with an amplicon size of 756 bp

Summary

Introduction

Molecular diagnostics aimed at the rapid detection of infectious diseases have become a powerful tool in modern medicine, with over 160 products currently approved by the FDA. An overwhelming majority of these tests are for clinical syndromes with very narrow aetiological spectrums, targeting single organisms or small, restricted panels of pathogens [1] Diagnosis of diseases such as human sepsis, where the causative agents in circulating blood display significant genetic diversity, presents a much greater challenge. 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 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

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