Optimizing an enclosed bead beating extraction method for microbial and fish environmental DNA

The environmental DNA (eDNA) method, which is widely applied for biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, Biryu-Chip (BC), and a filtration/extraction method, BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of the extracted DNA was obtained with 1/20–1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we could perform on-site measurements of eDNA within 30 min using a mobile PCR device. Using the BC method, filtration and extraction could be performed easily and quickly. The PCR results obtained by the BC method were similar to those obtained by the Sterivex method. The BC method required fewer steps and therefore, the risk of DNA contamination could be reduced. When combined with a mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from a few 10 mL of the water sample, even on-site.

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Rapid and affordable size-selected PacBio single-molecule real-time sequencing template library construction using the bead-beating DNA extraction method

Journal of Biological Methods ◽

10.14440/jbm.2017.169 ◽

2017 ◽

Vol 4 (3) ◽

pp. 79 ◽

Cited By ~ 2

Author(s):

Kengo Kato ◽

Masanori Hashino ◽

Tamaki Ito ◽

Mari Matsui ◽

Satowa Suzuki ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Single Molecule ◽

Extraction Method ◽

Library Construction ◽

Bead Beating ◽

Dna Extraction Method ◽

Template Library

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Universal extraction method for gastrointestinal pathogens

Journal of Medical Microbiology ◽

10.1099/jmm.0.058743-0 ◽

2013 ◽

Vol 62 (10) ◽

pp. 1535-1539 ◽

Cited By ~ 16

Author(s):

Fenella D. Halstead ◽

Adele V. Lee ◽

Xose Couto-Parada ◽

Spencer D. Polley ◽

Clare Ling ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Extraction Method ◽

Treatment Method ◽

Universal Method ◽

Rapid Diagnostics ◽

Time Saving ◽

Bead Beating ◽

Gastrointestinal Pathogens ◽

Disease Study

A universal stool extraction method for recovery of nucleic acids (NAs) from gastrointestinal pathogens was developed to support rapid diagnostics for the London 2012 Olympics. The method involved mechanical disruption (bead beating) of the stools, followed by automated extraction and detection using real-time PCR. This method had been used extensively in the Second Infectious Intestinal Disease Study (IID2) for the isolation of NA from bacteria and parasites (and was effective for the robust recovery of Cryptosporidium spp.) but had not been used for enteric viruses. To ensure this method was universally suitable, panels of samples known to contain target bacteria, viruses or parasites were processed in triplicate using the pre-treatment method routinely used for each target and the new extraction method (bead beating). The extracts were tested using real-time PCR and the cycle threshold values were compared. The results from this study showed that bead beating improved yields for the bacterial and parasitic targets and was suitable for the viral targets. The implementation of this universal method should confer cost- and time-saving benefits and streamline the processes required for the characterization of an array of pathogens from faecal samples.

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Filtration extraction method using microfluidic channel for measuring environmental DNA

10.1101/2021.11.18.468931 ◽

2021 ◽

Author(s):

Fukuzawa Takashi ◽

Yuichi Kameda ◽

Hisao Nagata ◽

Naofumi Nishizawa ◽

Hideyuki Doi

Keyword(s):

Water Sample ◽

River Water ◽

Dna Extraction ◽

Water Samples ◽

Extraction Method ◽

Microfluidic Channel ◽

Pcr Amplification ◽

Environmental Dna ◽

Laboratory Analysis ◽

River Water Samples

The environmental DNA (eDNA) method, which is widely applied for biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, Biryu-Chip (BC), and a filtration/extraction method, BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of the extracted DNA was obtained with 1/20-1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we could perform on-site measurements of eDNA within 30 min using a mobile PCR device. Using the BC method, filtration and extraction could be performed easily and quickly. The PCR results obtained by the BC method were similar to those obtained by the Sterivex method. However, the BC method required fewer steps and therefore, the risk of DNA contamination could be reduced. When combined with a mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from 10 mL of the water sample, even on-site.

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Storage media and not extraction method has the biggest impact on recovery of bacteria from the oral microbiome

Scientific Reports ◽

10.1038/s41598-019-51448-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Xiaoyan Zhou ◽

Shanika Nanayakkara ◽

Jin-Long Gao ◽

Ky-Anh Nguyen ◽

Christina J. Adler

Keyword(s):

Dna Extraction ◽

Dental Plaque ◽

Extraction Method ◽

Storage Conditions ◽

Oral Microbiome ◽

Rrna Gene ◽

Oral Microbiota ◽

Bead Beating ◽

Storage Media ◽

Dna Extraction Method

Abstract Next Generation sequencing has greatly progressed the exploration of the oral microbiome’s role in dental diseases, however, there has been little focus on the effect of sample storage conditions and their interaction with DNA extraction method. Dental plaque samples collected from 20 healthy participants were pooled and stored in either 75% ethanol or Bead solution for up to 6-months at −80 °C, prior to DNA extraction with either QIAamp (non-bead beating) or PowerSoil (bead-beating) kit, followed by Illumina sequencing of 16S rRNA gene. We found that storage media and not extraction method had the biggest influence on the diversity and abundance of the oral microbiota recovered. Samples stored in Bead solution, independent of the extraction kit, retrieved higher diversity (PowerSoil p = 1.64E-07, QIAamp p = 0.0085) and had dissimilar overall ecologies as indicated by lower level of shared diversity (PowerSoil p = 0.0000237, QIAamp p = 0.0088). Comparatively, samples stored in Bead solution and extracted with PowerSoil recovered a higher abundance of Streptococcus species. These data indicate that Bead solution can preserve the oral microbiome in dental plaque reliably, for periods of up to 6-months at −80 °C, and is compatible, with either a bead-beating or non-bead beating DNA extraction method.

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Sampling and Extraction Method for Environmental DNA (eDNA) in Freshwater Ecosystems

Korean Journal of Ecology and Environment ◽

10.11614/ksl.2021.54.3.170 ◽

2021 ◽

Vol 54 (3) ◽

pp. 170-189

Author(s):

Keonhee Kim ◽

Jeha Ryu ◽

Soon-jin Hwang

Keyword(s):

Extraction Method ◽

Environmental Dna ◽

Freshwater Ecosystems

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A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments

Frontiers in Microbiology ◽

10.3389/fmicb.2016.00986 ◽

2016 ◽

Vol 07 ◽

Cited By ~ 23

Author(s):

Vengadesh Perumal Natarajan ◽

Xinxu Zhang ◽

Yuki Morono ◽

Fumio Inagaki ◽

Fengping Wang

Keyword(s):

Dna Extraction ◽

Extraction Method ◽

Environmental Dna ◽

High Quality ◽

Dna Extraction Method

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Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method

Metabarcoding and Metagenomics ◽

10.3897/mbmg.2.28963 ◽

2018 ◽

Vol 2 ◽

Cited By ~ 18

Author(s):

Kristy Deiner ◽

Jacqueline Lopez ◽

Steve Bourne ◽

Luke Holman ◽

Mathew Seymour ◽

...

Keyword(s):

Pore Size ◽

Extraction Method ◽

False Negative ◽

Environmental Dna ◽

Isoamyl Alcohol ◽

Filter Material ◽

Extraction Methods ◽

Indigenous Species ◽

Taxonomic Richness ◽

Eukaryotic Dna

The analysis of environmental DNA (eDNA) using metabarcoding has increased in use as a method for tracking biodiversity of ecosystems. Little is known about eDNA in marine human-modified environments, such as commercial ports, which are key sites to monitor for anthropogenic impacts on coastal ecosystems. To optimise an eDNA metabarcoding protocol in these environments, seawater samples were collected in a commercial port and methodologies for concentrating and purifying eDNA were tested for their effect on eukaryotic DNA yield and subsequent richness of Operational Taxonomic Units (OTUs). Different filter materials [Cellulose Nitrate (CN) and Glass Fibre (GF)], with different pore sizes (0.5 µm, 0.7 µm and 1.2 µm) and three previously published liquid phase extraction methods were tested. The number of eukaryotic OTUs detected differed by a factor of three amongst the method combinations. The combination of CN filters with phenol-chloroform-isoamyl alcohol extractions recovered a higher amount of eukaryotic DNA and OTUs compared to GF filters and the chloroform-isoamyl alcohol extraction method. Pore size was not independent of filter material but did affect the yield of eukaryotic DNA. For the OTUs assigned to a highly successful non-indigenous species, Styelaclava, the two extraction methods with phenol significantly outperformed the extraction method without phenol; other experimental treatments did not contribute significantly to detection. These results highlight that careful consideration of methods is warranted because choice of filter material and extraction method create false negative detections of marine eukaryotic OTUs and underestimate taxonomic richness from environmental samples.

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