ArticlePloS one2015
Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 20 citations in OpenAlex.
- Application of Advanced Molecular Methods to Study Early-Onset Neonatal Sepsis.International journal of molecular sciences · 2024Review
- The diagnostic benefit of 16S rDNA PCR examination of infective endocarditis heart valves: a cohort study of 146 surgical cases confirmed by histopathology.Clinical research in cardiology : official journal of the German Cardiac Society · 2021Observational
- Evaluation of DNA extraction yield from a chlorinated drinking water distribution system.PloS one · 2021Article
- Increasing Reproducibility in Oral Microbiome Research.Methods in molecular biology (Clifton, N.J.) · 2021Article
- The Problem of Microbial Dark Matter in Neonatal Sepsis.Emerging infectious diseases · 2020Article
- Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories.Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc · 2020Article
- The challenge of recognising sepsis: Future nanotechnology solutions.Journal of the Intensive Care Society · 2020Review
- Article
- Lack of Evidence for Microbiota in the Placental and Fetal Tissues of Rhesus Macaques.mSphere · 2020Article
- The Madness of Microbiome: Attempting To Find Consensus "Best Practice" for 16S Microbiome Studies.Applied and environmental microbiology · 2018Review
- A novel approach to eliminate detection of contaminating Staphylococcal species introduced during clinical testing.PloS one · 2017Article
- Article
Corrections and comments
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Authors and funding
6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.