Comparison of inhibition resistance among PCR reagents for detection and quantification of environmental DNA

Uchii, Kimiko; Doi, Hideyuki; Okahashi, Teruyuki; Katano, Izumi; Yamanaka, Hiroki; Sakata, Masato; Minamoto, Toshifumi

doi:10.1002/edn3.37

Cited by 30 publications

(30 citation statements)

References 33 publications

(62 reference statements)

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“…Although ddPCR is robust against inhibition, high concentrations of PCR inhibitors like humic acids may also inhibit ddPCR. Several qPCR mastermixes are robust against inhibition, including Taqman Environmental Mastermix 2.0 (Strand et al, 2011;Uchii et al, 2019). Thus, the type of qPCR mastermix and its ability to deal with inhibition will highly influence the results in a comparison to ddPCR.…”

Section: Efficiency For Edna Detection Of Noble Crayfish -Qpcr Versusmentioning

confidence: 99%

Environmental DNA (eDNA) Monitoring of Noble Crayfish Astacus astacus in Lentic Environments Offers Reliable Presence-Absence Surveillance – But Fails to Predict Population Density

Johnsen

Strand

Rusch

et al. 2020

Front. Environ. Sci.

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Noble crayfish is the most widespread native freshwater crayfish species in Europe. It is threatened in its entire distribution range and listed on the International Union for Concervation Nature- and national red lists. Reliable monitoring data is a prerequisite for implementing conservation measures, and population trends are traditionally obtained from catch per unit effort (CPUE) data. Recently developed environmental DNA (eDNA) tools can potentially improve the effort. In the past decade, eDNA monitoring has emerged as a promising tool for species surveillance, and some studies have established that eDNA methods yield adequate presence-absence data for crayfish. There are also high expectations that eDNA concentrations in the water can predict biomass or relative density. However, eDNA studies for crayfish have not yet been able to establish a convincing relationship between eDNA concentrations and crayfish density. This study compared eDNA and CPUE data obtained the same day and with high sampling effort, and evaluated whether eDNA concentrations can predict relative density of crayfish. We also compared two analytical methods [Quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR)], and estimated the detection probability for eDNA monitoring compared to trapping using occupancy modeling. In all lakes investigated, we detected eDNA from noble crayfish, even in lakes with very low densities. The eDNA method is reliable for presence-absence monitoring of noble crayfish, and the probability of detecting noble crayfish from eDNA samples increased with increasing relative crayfish densities. However, the crayfish eDNA concentrations were consistently low and mostly below the limit of quantification, even in lakes with very high crayfish densities. The hypothesis that eDNA concentrations can predict relative crayfish density was consequently not supported. Our study underlines the importance of intensified sampling effort for successful detection of very low-density populations, and for substantiating presumed absence, inferred from negative results. Surprisingly, we found a higher likelihood of eDNA detection using qPCR compared to ddPCR. We conclude that eDNA monitoring cannot substitute CPUE data, but is a reliable supplement for rapid presence-absence overviews. Combined with eDNA analyses of alien crayfish species and diseases such as crayfish plague, this is a cost-efficient supplement offering a more holistic monitoring approach for aquatic environments and native crayfish conservation.

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Section: Efficiency For Edna Detection Of Noble Crayfish -Qpcr Versusmentioning

confidence: 99%

Environmental DNA (eDNA) Monitoring of Noble Crayfish Astacus astacus in Lentic Environments Offers Reliable Presence-Absence Surveillance – But Fails to Predict Population Density

Johnsen

Strand

Rusch

et al. 2020

Front. Environ. Sci.

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“…Most eDNA studies that use a filtration approach have been conducted in marine or freshwater systems, where water appears to be non-turbid at the time of collection [15][16][17][18]. This is owing to the unique set of challenges that turbid water poses when detecting eDNA, such as the clogging of filters and the presence of PCR inhibitors [19][20][21]. Previous studies have utilised extraction kits-which come with anti-inhibitory washes-various pore sizes, membrane types, and pre-filtration steps to prevent filter clogging [22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding

Takasaki¹,

Aihara²,

Imanaka³

et al. 2021

PLoS ONE

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Environmental DNA (eDNA) analysis is a novel approach for biomonitoring and has been mostly used in clear water. It is difficult to detect eDNA in turbid water as filter clogging occurs, and environmental samples contain various substances that inhibit the polymerase chain reaction (PCR) and affect the accuracy of eDNA analysis. Therefore, we applied a pre-filtration method to better detect the fish species (particularly pale chub, Opsariichthys platypus) present in a water body by measuring eDNA in environmental samples containing PCR inhibitors. Upon conducting 12S rRNA metabarcoding analysis (MiFish), we found that pre-filtration did not affect the number or identities of fish species detected in our samples, but pre-filtration through pore sizes resulted in significantly reduced variance among replicate samples. Additionally, PCR amplification was improved by the pre-filtration of environmental samples containing PCR inhibitors such as humic substances. Although this study may appear to be a conservative and ancillary experiment, pre-filtration is a simple technique that can not only improve the physical properties of water, such as turbidity, but also the quality of eDNA biomonitoring.

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“…Our group previously demonstrated the importance of purification (PowerClean Pro Cleanup kit) on the detection of chum salmon eDNA from the field samples as they carried enormous amount of PCR inhibitors that compromise the qPCR reaction 5,25 . However, among new formulations of PCR reagents that are becoming available on the markets, some are designed to have high tolerance to PCR inhibitors in environmental samples 26 . In our previous study 5 , the ABI Environmental Master Mix 2.0 was robust to perform qPCR on environmental samples purified by PowerClean kit.…”

Section: Discussionmentioning

confidence: 99%

“…4). Various types of PCR reagents have different degree of tolerance against known PCR inhibitors including humic, fulvic, and tannic acids 26 and the use of Takara Probe qPCR Mix could be advantageous specifically to our sampling sites. We explored the possibility of whether the eDNA can be quantified directly after DNeasy extraction without additional purification by the PowerClean kit.…”

Section: Discussionmentioning

confidence: 99%

Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter

Wong

Nakao

Hyodo

2020

Sci Rep

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Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of the samples. We improved the eDNA extraction efficiency of an established Sterivex method by 12- to 16-fold using a larger volume of lysis buffer mix coupled with backflushing the cartridges. The DNeasy extraction column could be overloaded when the environmental sample input is high, possibly due to a higher nonspecific binding present in environmental samples, thus resulting in a relatively lower quantity measured. Therefore, we included an internal control DNA in the extraction to monitor the extraction and purification efficiencies in field samples, which is crucial for quantification of original eDNA concentration. The use of Takara Probe qPCR Mix supplemented with protein-based additives improved the robustness of the real time PCR assay on inhibitor-rich environmental samples, but prior purification by Qiagen PowerClean Pro Cleanup kit could be essential for inhibitor-rich water samples, even though the recovery rate was unexpectedly low (average 33.0%). The improved extraction and quantification complement the qualitative analyses including metabarcoding and metagenomics in field application.

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Comparison of inhibition resistance among PCR reagents for detection and quantification of environmental DNA

Cited by 30 publications

References 33 publications

Environmental DNA (eDNA) Monitoring of Noble Crayfish Astacus astacus in Lentic Environments Offers Reliable Presence-Absence Surveillance – But Fails to Predict Population Density

Environmental DNA (eDNA) Monitoring of Noble Crayfish Astacus astacus in Lentic Environments Offers Reliable Presence-Absence Surveillance – But Fails to Predict Population Density

Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding

Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter

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