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Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

Turner CR, Miller DJ, Coyne KJ, Corush J - PLoS ONE (2014)

Bottom Line: As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species.We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols.Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States of America.

ABSTRACT
Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

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qPCR assay results comparing the amount of bigheaded carp eDNA captured and recovered (i.e., eDNA yield) using two alternative capture/extraction methods on paired 2 L samples collected side-by-side in the experimental pond at USGS-CERC.48 pairs of samples were collected, but following Zuur et al. [88], seven pairs with unusually high eDNA concentration (i.e., outliers) were removed prior to statistical analysis. Statistical results were robust to outlier presence or removal, and plots including outliers are provided in Figure S1. PCTE = polycarbonate track-etched filter membrane, GF = glass fiber filter paper, CTAB = cetyl trimethyl ammonium bromide DNA extraction protocol, PowerWater = PowerWater DNA Isolation Kit. (A) Paired data, (B) boxplot, (C) paired differences and the median difference (red point) and 95% confidence interval (red interval) from the Wilcoxon signed-rank test. Note that points in (C) are horizontally ‘jittered’ for better visualization. The PCTE/CTAB method yielded significantly more eDNA than the GF/PowerWater method (paired Student’s t-test, t = 4.1, df = 40, P = 0.00019).
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pone-0114329-g002: qPCR assay results comparing the amount of bigheaded carp eDNA captured and recovered (i.e., eDNA yield) using two alternative capture/extraction methods on paired 2 L samples collected side-by-side in the experimental pond at USGS-CERC.48 pairs of samples were collected, but following Zuur et al. [88], seven pairs with unusually high eDNA concentration (i.e., outliers) were removed prior to statistical analysis. Statistical results were robust to outlier presence or removal, and plots including outliers are provided in Figure S1. PCTE = polycarbonate track-etched filter membrane, GF = glass fiber filter paper, CTAB = cetyl trimethyl ammonium bromide DNA extraction protocol, PowerWater = PowerWater DNA Isolation Kit. (A) Paired data, (B) boxplot, (C) paired differences and the median difference (red point) and 95% confidence interval (red interval) from the Wilcoxon signed-rank test. Note that points in (C) are horizontally ‘jittered’ for better visualization. The PCTE/CTAB method yielded significantly more eDNA than the GF/PowerWater method (paired Student’s t-test, t = 4.1, df = 40, P = 0.00019).

Mentions: In testing at the USGS-CERC experimental pond, detection probability was not significantly different between eDNA capture/extraction methods for either type of assay (endpoint PCR or qPCR; McNemar's χ2 = 2.25, df = 1, P = 0.13, McNemar's χ2 = 0, df = 1, P = 1; Table 2) but the PCTE filter and CTAB extraction method yielded five times more bigheaded carp eDNA, on average, than the GF filter and PowerWater extraction (paired Student’s t-test, t = 4.1, df = 40, P = 0.00019; Figure 2). The 5-fold higher yield may reflect higher eDNA capture, extraction recovery, or both. However, the larger effective [69], [70] and nominal pore size of the PCTE filter (10 µm vs. 1.5 µm for GF) suggests eDNA capture would be higher for the GF filter. This points to extraction recovery as the more important process influencing final yield in our comparison, which is expected because phase separation and precipitation methods (e.g., CTAB) consistently yield more DNA than silica column methods (e.g., PowerWater) [71], [72]. Thus we recommend the CTAB extraction method for general application in eDNA-based monitoring. Its efficacy has been demonstrated on a wide range of environmental samples, from aquatic sediments [43], [73] and volcanic rock [74] to retentate on various types of filter material including PCTE [44], polyethersulfone (PES) [75], cellulose nitrate (nitrocellulose, pyroxylin) [76], cellulose acetate [77], glass fiber [78], and nylon net [79]. During the CTAB extraction, chloroform dissolves PCTE [80] and PES filters [81], facilitating the recovery of any DNA-containing particles embedded in the filter matrix [67], [82]. This chemical dissolution also enables simple scale up of the extraction volume to accommodate multiple filters or large surface area filters. Finally, for eDNA capture in the field, immediately storing a used filter in CTAB buffer (Protocol S1) reduces DNA degradation [83]–[85] because CTAB lyses cells while EDTA and salt inactivate nucleases.


Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

Turner CR, Miller DJ, Coyne KJ, Corush J - PLoS ONE (2014)

qPCR assay results comparing the amount of bigheaded carp eDNA captured and recovered (i.e., eDNA yield) using two alternative capture/extraction methods on paired 2 L samples collected side-by-side in the experimental pond at USGS-CERC.48 pairs of samples were collected, but following Zuur et al. [88], seven pairs with unusually high eDNA concentration (i.e., outliers) were removed prior to statistical analysis. Statistical results were robust to outlier presence or removal, and plots including outliers are provided in Figure S1. PCTE = polycarbonate track-etched filter membrane, GF = glass fiber filter paper, CTAB = cetyl trimethyl ammonium bromide DNA extraction protocol, PowerWater = PowerWater DNA Isolation Kit. (A) Paired data, (B) boxplot, (C) paired differences and the median difference (red point) and 95% confidence interval (red interval) from the Wilcoxon signed-rank test. Note that points in (C) are horizontally ‘jittered’ for better visualization. The PCTE/CTAB method yielded significantly more eDNA than the GF/PowerWater method (paired Student’s t-test, t = 4.1, df = 40, P = 0.00019).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256254&req=5

pone-0114329-g002: qPCR assay results comparing the amount of bigheaded carp eDNA captured and recovered (i.e., eDNA yield) using two alternative capture/extraction methods on paired 2 L samples collected side-by-side in the experimental pond at USGS-CERC.48 pairs of samples were collected, but following Zuur et al. [88], seven pairs with unusually high eDNA concentration (i.e., outliers) were removed prior to statistical analysis. Statistical results were robust to outlier presence or removal, and plots including outliers are provided in Figure S1. PCTE = polycarbonate track-etched filter membrane, GF = glass fiber filter paper, CTAB = cetyl trimethyl ammonium bromide DNA extraction protocol, PowerWater = PowerWater DNA Isolation Kit. (A) Paired data, (B) boxplot, (C) paired differences and the median difference (red point) and 95% confidence interval (red interval) from the Wilcoxon signed-rank test. Note that points in (C) are horizontally ‘jittered’ for better visualization. The PCTE/CTAB method yielded significantly more eDNA than the GF/PowerWater method (paired Student’s t-test, t = 4.1, df = 40, P = 0.00019).
Mentions: In testing at the USGS-CERC experimental pond, detection probability was not significantly different between eDNA capture/extraction methods for either type of assay (endpoint PCR or qPCR; McNemar's χ2 = 2.25, df = 1, P = 0.13, McNemar's χ2 = 0, df = 1, P = 1; Table 2) but the PCTE filter and CTAB extraction method yielded five times more bigheaded carp eDNA, on average, than the GF filter and PowerWater extraction (paired Student’s t-test, t = 4.1, df = 40, P = 0.00019; Figure 2). The 5-fold higher yield may reflect higher eDNA capture, extraction recovery, or both. However, the larger effective [69], [70] and nominal pore size of the PCTE filter (10 µm vs. 1.5 µm for GF) suggests eDNA capture would be higher for the GF filter. This points to extraction recovery as the more important process influencing final yield in our comparison, which is expected because phase separation and precipitation methods (e.g., CTAB) consistently yield more DNA than silica column methods (e.g., PowerWater) [71], [72]. Thus we recommend the CTAB extraction method for general application in eDNA-based monitoring. Its efficacy has been demonstrated on a wide range of environmental samples, from aquatic sediments [43], [73] and volcanic rock [74] to retentate on various types of filter material including PCTE [44], polyethersulfone (PES) [75], cellulose nitrate (nitrocellulose, pyroxylin) [76], cellulose acetate [77], glass fiber [78], and nylon net [79]. During the CTAB extraction, chloroform dissolves PCTE [80] and PES filters [81], facilitating the recovery of any DNA-containing particles embedded in the filter matrix [67], [82]. This chemical dissolution also enables simple scale up of the extraction volume to accommodate multiple filters or large surface area filters. Finally, for eDNA capture in the field, immediately storing a used filter in CTAB buffer (Protocol S1) reduces DNA degradation [83]–[85] because CTAB lyses cells while EDTA and salt inactivate nucleases.

Bottom Line: As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species.We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols.Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States of America.

ABSTRACT
Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

Show MeSH