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

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.

The Madness of Microbiome: Attempting To Find Consensus “Best Practice” for 16S Microbiome Studies

“…The amplification of background contaminants from PCR reagents could perhaps be avoided via the use of primer-extension PCR ( 102 ), but this would have no effect on contamination originating from other sources. Several methods have been suggested to remove contaminating DNA from reagents and the lab environment, including UV and gamma radiation ( 103 – 107 ); DNA intercalation by 8-methoxypsoralen, ethidium monoazide, and propidium monoazide ( 104 , 106 – 108 ); enzymatic treatments ( 105 – 107 , 109 – 111 ); silica-based membrane filtration ( 112 ); CsCl 2 density gradient centrifugation ( 111 ); and bleach/copper-bis-(phenanthroline)-sulfate/H 2 O 2 (CoPA) solution treatment ( 105 ). These methods have shown varied effects on contamination levels and PCR sensitivity, and the inclusion of reagent-only controls alongside these decontamination measures is still recommended.…”

From Lab to Lake – Evaluation of Current Molecular Methods for the Detection of Infectious Enteric Viruses in Complex Water Matrices in an Urban Area

“…Both reagents contain a photo-inducible azide group that covalently binds to nucleic acids after exposure to light with a specific wavelength which results in a significantly decreased signal in a subsequent qPCR due to the inhibition of the polymerase [13]. The usage of PMA and EMA has been proposed for the selective detection of a broad spectrum of organisms including bacteria [14–18], fungi [19, 20], various protozoa including incorporated bacteria [21–23] and nematode eggs [24]. The application of the method for the distinction between infectious and non-infectious viruses has been investigated thoroughly in lab scale [25–29].…”

Lack of evidence for microbiota in the placental and fetal tissues of rhesus macaques

“…Pelomonas was not reported in prior preliminary molecular surveys of the fetal and placental tissues of macaques (85, 89), and it has only been reported in a single study as being a component of the human placental microbiota (41). Yet, Pelomonas has been identified as a background DNA contaminant in sequence-based studies (47, 48, 94), including in prior studies of the human placenta (57, 59, 62). As with Staphylococcus , in the current study, the prominent ASV classified as Pelomonas was also prominent and widespread among the background technical control samples, suggesting that it was a background DNA contaminant.…”