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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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Alignment diagram showing the relative position of oligonucleotides for the qPCR and endpoint PCR assays.The qPCR assay was developed in the present study and the endpoint PCR assays are from a previous study [40]. Gaps (−) and bases in between oligonucleotides (… n bp…) reflect the actual alignment used to design the qPCR assay. Note that all oligonucleotides are shown on the ‘sense’ strand in 5′ to 3′ orientation, left to right. Thus oligonucleotides that actually bind to the sense strand (i.e., reverse primers and the hydrolysis probe) are shown as reverse-complements of the actual oligonucleotides used in an assay (Table 1). Degenerate bases are shown in bold text, but oligonucleotides were not synthesized with degenerate bases for the qPCR assay (Table 1). The forward primer of the Silver Carp endpoint PCR assay was synthesized with degenerate bases, as specified in the study that developed it [40].
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pone-0114329-g001: Alignment diagram showing the relative position of oligonucleotides for the qPCR and endpoint PCR assays.The qPCR assay was developed in the present study and the endpoint PCR assays are from a previous study [40]. Gaps (−) and bases in between oligonucleotides (… n bp…) reflect the actual alignment used to design the qPCR assay. Note that all oligonucleotides are shown on the ‘sense’ strand in 5′ to 3′ orientation, left to right. Thus oligonucleotides that actually bind to the sense strand (i.e., reverse primers and the hydrolysis probe) are shown as reverse-complements of the actual oligonucleotides used in an assay (Table 1). Degenerate bases are shown in bold text, but oligonucleotides were not synthesized with degenerate bases for the qPCR assay (Table 1). The forward primer of the Silver Carp endpoint PCR assay was synthesized with degenerate bases, as specified in the study that developed it [40].

Mentions: A set of candidate primers and probes was selected for in vitro testing with 1 ng•µL−1 standardized tissue-derived total genomic DNA from H. nobilis, H. molitrix, C. carpio, C. auratus, and C. idella. Importantly, obtaining uncontaminated samples of tissue from these closely related, co-occurring species required considerable and repeated effort, and we recommend extreme care to prevent cross-contamination of assay validation samples during collection, handling, transport, storage, extraction, etc. qPCR assays targeting low level DNA are notoriously sensitive to contamination, and their use requires extra precautions that are uncommon in ecological research and even in standard genetics laboratories [49], [50]. We selected one primer set (100 bp amplicon) and one MGB probe (Table 1, Figure 1) that demonstrated identical amplification efficiency from both H. nobilis and H. molitrix DNA and no amplification from any of the nontarget species’ DNA. Note that we designed two slightly different versions of the forward and reverse primers (Table 1), reflecting three positions that vary within bigheaded carp, according to all available GenBank sequences. Rather than ordering primers with degenerate bases, which would create unnecessary combinations of the two variable positions in the reverse primer, we simply ordered each version separately and combined them at equimolar concentration. We also designed and tested a Locked Nucleic Acid (LNA; Sigma-Aldrich, St. Louis, Missouri, USA) [51] version of the probe (Table 1) and found equivalent performance (data not shown). All results in this study were generated using the MGB version of the probe.


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)

Alignment diagram showing the relative position of oligonucleotides for the qPCR and endpoint PCR assays.The qPCR assay was developed in the present study and the endpoint PCR assays are from a previous study [40]. Gaps (−) and bases in between oligonucleotides (… n bp…) reflect the actual alignment used to design the qPCR assay. Note that all oligonucleotides are shown on the ‘sense’ strand in 5′ to 3′ orientation, left to right. Thus oligonucleotides that actually bind to the sense strand (i.e., reverse primers and the hydrolysis probe) are shown as reverse-complements of the actual oligonucleotides used in an assay (Table 1). Degenerate bases are shown in bold text, but oligonucleotides were not synthesized with degenerate bases for the qPCR assay (Table 1). The forward primer of the Silver Carp endpoint PCR assay was synthesized with degenerate bases, as specified in the study that developed it [40].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256254&req=5

pone-0114329-g001: Alignment diagram showing the relative position of oligonucleotides for the qPCR and endpoint PCR assays.The qPCR assay was developed in the present study and the endpoint PCR assays are from a previous study [40]. Gaps (−) and bases in between oligonucleotides (… n bp…) reflect the actual alignment used to design the qPCR assay. Note that all oligonucleotides are shown on the ‘sense’ strand in 5′ to 3′ orientation, left to right. Thus oligonucleotides that actually bind to the sense strand (i.e., reverse primers and the hydrolysis probe) are shown as reverse-complements of the actual oligonucleotides used in an assay (Table 1). Degenerate bases are shown in bold text, but oligonucleotides were not synthesized with degenerate bases for the qPCR assay (Table 1). The forward primer of the Silver Carp endpoint PCR assay was synthesized with degenerate bases, as specified in the study that developed it [40].
Mentions: A set of candidate primers and probes was selected for in vitro testing with 1 ng•µL−1 standardized tissue-derived total genomic DNA from H. nobilis, H. molitrix, C. carpio, C. auratus, and C. idella. Importantly, obtaining uncontaminated samples of tissue from these closely related, co-occurring species required considerable and repeated effort, and we recommend extreme care to prevent cross-contamination of assay validation samples during collection, handling, transport, storage, extraction, etc. qPCR assays targeting low level DNA are notoriously sensitive to contamination, and their use requires extra precautions that are uncommon in ecological research and even in standard genetics laboratories [49], [50]. We selected one primer set (100 bp amplicon) and one MGB probe (Table 1, Figure 1) that demonstrated identical amplification efficiency from both H. nobilis and H. molitrix DNA and no amplification from any of the nontarget species’ DNA. Note that we designed two slightly different versions of the forward and reverse primers (Table 1), reflecting three positions that vary within bigheaded carp, according to all available GenBank sequences. Rather than ordering primers with degenerate bases, which would create unnecessary combinations of the two variable positions in the reverse primer, we simply ordered each version separately and combined them at equimolar concentration. We also designed and tested a Locked Nucleic Acid (LNA; Sigma-Aldrich, St. Louis, Missouri, USA) [51] version of the probe (Table 1) and found equivalent performance (data not shown). All results in this study were generated using the MGB version of the probe.

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