Development of an ethidium monoazide–enhanced internally controlled universal 16S rDNA real‐time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates

Patel, Poorvi; Garson, Jeremy A.; Tettmar, Kate I.; Ancliff, S.; McDonald, Carl; Pitt, T. L.; Coelho, Juliana; Tedder, Richard S.

doi:10.1111/j.1537-2995.2011.03484.x

Cited by 37 publications

(58 citation statements)

References 30 publications

(64 reference statements)

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“…A number of methods have been devised to treat reagents in order to reduce potential contamination, including: gamma [45] or UV radiation [13,46–48], DNase treatment [10,13,47,49–51], restriction digests [10,13,47,52,53], caesium chloride density gradient centrifugation [10] and DNA intercalation and crosslinking with 8-methoxypsoralen [47,54], propidium monoazide [55] or ethidium monoazide [56,57]. However, tests of these methods show varying levels of success.…”

Section: Discussionmentioning

confidence: 99%

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

et al. 2014

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BackgroundThe study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents.ResultsIn this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination.ConclusionsThese results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0087-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

et al. 2014

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“…from Thermus aquaticus bacteria used to synthesise DNA Taq DNA polymerase). A number of approaches have been proposed for the removal of contaminating DNA from PCR reaction solutions including treatment with UV light (Ou et al 1991), restriction endonucleases (Carroll et al 1999) and ethidium monoazide (Patel et al 2012), with various success. Certified DNA-free polymerases are available for purchase (e.g.…”

Section: Use Of Appropriate Pcr Controlsmentioning

confidence: 99%

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Lear¹,

Dickie²,

Banks³

et al. 2018

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Advances in the sequencing of DNA extracted from media such as soil and water offer huge opportunities for biodiversity monitoring and assessment, particularly where the collection or identification of whole organisms is impractical. However, there are myriad methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. To help overcome potential biases that may impede the effective comparison of biodiversity data collected by different researchers, we propose a standardised set of procedures for use on different taxa and sample media, largely based on recent trends in their use. Our recommendations describe important steps for sample pre-processing and include the use of (a) Qiagen DNeasy PowerSoil ® and PowerMax ® kits for extraction of DNA from soil, sediment, faeces and leaf litter; (b) DNeasy PowerSoil ® for extraction of DNA from plant tissue; (c) DNeasy Blood and Tissue kits for extraction of DNA from animal tissue; (d) DNeasy Blood and Tissue kits for extraction of DNA from macroorganisms in water and ice; and (e) DNeasy PowerWater ® kits for extraction of DNA from microorganisms in water and ice. Based on key parameters, including the specificity and inclusivity of the primers for the target sequence, we recommend the use of the following primer pairs to amplify DNA for analysis by Illumina MiSeq DNA sequencing: (a) 515f and 806RB to target bacterial 16S rRNA genes (including regions V3 and V4); (b) #3 and #5RC to target eukaryote 18S rRNA genes (including regions V7 and V8); (c) #3 and #5RC are also recommended for the routine analysis of protist community DNA; (d) ITS6F and ITS7R to target the chromistan ITS1 internal transcribed spacer region; (e) S2F and S3R to target the ITS2 internal transcribed spacer in terrestrial plants; (f) fITS7 or gITS7, and ITS4 to target the fungal ITS2 region; (g) NS31 and AML2 to target glomeromycota 18S rRNA genes; and (h) mICOIintF and jgHCO2198 to target cytochrome c oxidase subunit I (COI) genes in animals. More research is currently required to confirm primers suitable for the selective amplification of DNA from specific vertebrate taxa such as fish. Combined, these recommendations represent a framework for efficient, comprehensive and robust DNA-based investigations of biodiversity, applicable to most taxa and ecosystems. The adoption of standardised protocols for biodiversity assessment and monitoring using DNA extracted from environmental samples will enable more informative comparisons among datasets, generating significant benefits for ecological science and biosecurity applications.

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“…On the other hand, it is known that MPRCA and MDA are easily affected by exogenous contamination of DNA, as is often observed in the polymerase chain reaction (PCR) [33]–[53]. In particular, any DNA amplification in non-template control (NTC) does not ensure proper MDA reaction in test samples taking place in parallel 54,55.…”

Section: Introductionmentioning

confidence: 99%

“…Various strategies aimed at eliminating contaminating DNA in PCR reagents have been reported and discussed [33]–[53], including DNase I treatment and short-wavelength ultraviolet (UV-C) irradiation; the latter strategy has also been used to prepare amplifiable DNA-free Phi29 DNA polymerase [25], [59], [60]. However, the polymerase itself may protect the contaminating DNA, perhaps captured by the polymerase, against DNase I in procedures such as DNase footprinting [47], [65].…”

Section: Introductionmentioning

confidence: 99%

Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

et al. 2014

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We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

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Development of an ethidium monoazide–enhanced internally controlled universal 16S rDNA real‐time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates

Cited by 37 publications

References 30 publications

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

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