title: Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
authors.affiliation: Institute of Inflammation and Repair, Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom; Infection, Injury & inflammation Research Group, Salford Royal NHS Foundation Trust, Salford, United Kingdom.
authors.forename: Bruce
authors.initials: B
authors.lastname: Humphrey
authors.affiliation: Public Health England, Microbiology Services Division, Porton Down, Salisbury, United Kingdom.
authors.forename: Neil
authors.initials: N
authors.lastname: McLeod
authors.affiliation: Public Health England, Microbiology Services Division, Porton Down, Salisbury, United Kingdom.
authors.forename: Carrie
authors.initials: C
authors.lastname: Turner
authors.affiliation: Public Health England, Microbiology Services Division, Porton Down, Salisbury, United Kingdom.
authors.forename: J Mark
authors.initials: JM
authors.lastname: Sutton
authors.affiliation: Institute of Inflammation and Repair, Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom; Infection, Injury & inflammation Research Group, Salford Royal NHS Foundation Trust, Salford, United Kingdom.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Dark
authors.affiliation: Institute of Inflammation and Repair, Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom; Infection, Injury & inflammation Research Group, Salford Royal NHS Foundation Trust, Salford, United Kingdom.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Warhurst
mesh_terms: D001386:Azides / Q000737:chemistry*; D059018:DNA Contamination; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics*; D003666:Decontamination / Q000379:methods*; D018628:Gene Dosage / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008969:Molecular Sequence Data; D011419:Propidium / Q000031:analogs & derivatives / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
chemical_list: D001386:Azides; D017931:DNA Primers; D004269:DNA, Bacterial; D007202:Indicators and Reagents; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium