Development of quantitative PCR assays for the detection and quantification of lake sturgeon (Acipenser fulvescens) environmental DNA

Yusishen, Michael E.; Eichorn, Frances-Claire; Anderson, W. Gary; Docker, Margaret F.

doi:10.1007/s12686-018-1054-8

Cited by 16 publications

(13 citation statements)

References 16 publications

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“…The number of eDNA studies has expanded exponentially in recent years. Some researchers make claims about primer specificity based soley on in silico validation (e.g., Wei et al 2018) or in combination with in vitro tests of one or a few nontarget species (e.g., Yusishen et al 2020). These practices are not recommended since, as our results show, in silico testing of primer specificity using BLAST can be inaccurate.…”

Section: Discussionmentioning

confidence: 99%

Pitfalls during in silico prediction of primer specificity for eDNA surveillance

Fong²,

Lam³

et al. 2020

Ecosphere

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While high efficiency and cost‐effectiveness are two merits of environmental DNA (eDNA) techniques for detecting aquatic organisms, the difficulty of designing species‐specific primers can result in significant expenditure of time and money. During the in silico stage of primer development, primer specificity is predicted with alignment techniques such as BLAST that is based on the number and position of the primer/nontarget template mismatches. However, we speculate that nonspecific amplification is influenced by additional parameters, which lead to inaccuracies of in silico prediction. We performed in vitro specificity tests for 38 species‐specific primers selected for seven fishes and six turtles, using single‐plex conventional PCR (cPCR). A subset of 12 primer pairs were further tested with SYBR Green‐based or TaqMan‐based single‐plex quantitative PCR (qPCR). We disentangle the relative importance of mismatch properties (types and positions), primer properties (length, GC content, and 3′ end stability), PCR conditions (template concentrations and annealing temperatures), and PCR technique (cPCR, TaqMan‐based, or SYBR Green‐based qPCR) in determining the occurrence of amplifications. We then compared the PCR outcomes with the specificity check under two stringency scenarios based on alignment (i.e., BLAST search). We conducted a total of 679 cPCR and 226 qPCR analyses, with 90% of the reactions tested with nontarget templates. Primer pairs predicted by Primer‐BLAST to be specific rarely showed such specificity during the in vitro testing. BLAST searches correctly predicted the outcomes of around 67% of cPCR and qPCR, but had low sensitivity in detection of nontarget amplification (29–57%). Primer specificity increased significantly with total number of mismatches and annealing temperature, but decreased with higher GC content in the primer sequence. Mismatches that consisted of A‐A, G‐A, and C‐C pairings exerted 56% stronger reduction in nonspecific amplification effects than other mismatches. To conclude, we show that the prediction of primer specificity based only on the number and position of mismatches can be misleading. Our findings can be applied to increase the efficiency of the in silico primer selection process to maintain the relatively high efficiency and cost‐effectiveness of eDNA techniques.

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Section: Discussionmentioning

confidence: 99%

Pitfalls during in silico prediction of primer specificity for eDNA surveillance

Fong²,

Lam³

et al. 2020

Ecosphere

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“…Irrespective of the various factors affecting the concentration of eDNA in an aquatic system, subsequently estimating a population's abundance or biomass is contingent on the repeatability of the eDNA concentration estimate. This estimate is the product of the compounding effects of many processes including the substrate used (water/sediment) and method of eDNA capture, extraction, storage, eDNA primer design and validation, and PCR amplification (Baldigo et al, 2017;Eichmiller et al, 2016;Furlan et al, 2016;Hinlo et al, 2017b;Jane et al, 2015;Knudsen et al, 2019;Kumar et al, 2020;Nathan et al, 2014;Thalinger et al, 2020;Thomsen et al, 2016;Yusishen et al, 2020). In the reviewed papers, most studies used a filtration method (e.g., glass fiber filters, mixed cellulose ester filters) to capture eDNA.…”

Section: Methods Of Capturing Extracting and Amplifying Ednamentioning

confidence: 99%

Environmental DNA (eDNA) as a tool for assessing fish biomass: A review of approaches and future considerations for resource surveys

et al. 2021

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Environmental DNA (eDNA) has revolutionized our ability to identify the presence and distributions of terrestrial and aquatic organisms. Recent evidence suggests the concentration of eDNA could also provide a rapid, cost-effective indicator of abundance and/or biomass for fisheries stock assessments. Globally, fisheries resources are under immense pressure, and their sustainable harvest requires accurate information on the sizes of fished stocks. However, in many cases the required information remains elusive because of a reliance on imprecise or costly fishery-dependent and independent data. Here, we review the literature describing relationships between eDNA concentrations and fish abundance and/or biomass, as well as key influencing factors, as a precursor to determining the broader utility of eDNA for monitoring fish populations.We reviewed 63 studies published between 2012 and 2020 and found 90% identified positive relationships between eDNA concentrations and the abundance and/or biomass of focal species. Key influencing biotic factors included the taxon examined as well as their body size, distribution, reproduction, and migration. Key abiotic factors mostly comprised hydrological processes affecting the dispersal and persistence of eDNA, especially water flow and temperature, although eDNA collection methods were also influential. The cumulative influence of these different factors likely explains the substantial variability observed in eDNA concentrations, both within and among studies. Nevertheless, there is considerable evidence to support using eDNA as an ancillary tool for assessing fish population abundance and/or biomass across discrete spatio-temporal scales, following preliminary investigations to determine speciesand context-specific factors influencing the eDNA abundance/biomass relationship.Advantages of eDNA monitoring relative to other approaches include reduced costs, increased efficiencies, and nonlethal sampling.

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“…In addition, a significant correlation has been observed between species relative abundance and the number of standardized reads, enabling a quantitative estimate of fish assemblage structures (hereafter referred to as relative species abundance) (Pont et al 2018;Rourke et al 2022).On the other hand, there are some limitations in the applicability and informative power of the method, such as false negatives and false positives, lack of information on population structure or the exact location of a sturgeon (see also Discussion). To date, sturgeons have been targeted only in a very limited number of eDNA studies in the USA (Mobile River Basin (Pfleger et al 2016), Sacramento River (Bergman et al 2016), and Hudson River (Stoeckle et al 2017)), Canada [Winnipeg River (Yusishen et al 2020)], and China [Yangtze River (Xu et al 2018)]. In contrast, there have been no eDNA-based studies conducted in the River Danube for any of its native species.…”

Section: Introductionmentioning

confidence: 99%

Sturgeons in large rivers: detecting the near-extinct needles in a haystack via eDNA metabarcoding from water samples

et al. 2022

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Sturgeon populations are declining worldwide and are the target of extensive conservation efforts. Addressed in several pieces of legislation, sturgeons have received considerable attention as flagship or umbrella species. Despite the need for a better understanding of the distribution and population status, the use of traditional sampling methods failed in the past, thereby hampering reliable assessments, a prerequisite for conservation. Here, we describe the development and application of an environmental DNA (eDNA) metabarcoding approach for detecting rare sturgeons in large rivers. Exemplarily, we developed a reference database for five native Danube sturgeons (Acipenser stellatus, Acipenser gueldenstaedtii, Acipenser ruthenus, Acipenser nudiventris, and Huso huso) and two non-native species (Acipenser baerii and Acipenser transmontanus), assessed these ex situ, and used eDNA as a detection tool along the entire length of the Danube (Europe, ~ 2850 km) and major tributaries. In ex situ analyses, all assays yielded positive amplifications for the assessed sturgeon species. In the Danube, the presence of A. ruthenus was confirmed at 14 of 29 sites (48.3%), and in 2 of 18 tributary sites (11.1%), providing the first comprehensive large-scale biogeographical snapshot of this species. Relative number of reads assigned to A. ruthenus varied between 0 and 2.5%, with sites registering positive detections being clustered in 3 sections of the Danube. Our findings enabled us to confirm the advantages of eDNA monitoring over traditional sampling methods for comprehensive whole-river snapshot studies of sturgeons conducted on a large geographical scale, and therefore we consider it to be a promising approach for application in conservation measures, fisheries management, scientific studies, and adaptive management plans for sturgeons on a global scale.

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Development of quantitative PCR assays for the detection and quantification of lake sturgeon (Acipenser fulvescens) environmental DNA

Cited by 16 publications

References 16 publications

Pitfalls during in silico prediction of primer specificity for eDNA surveillance

Pitfalls during in silico prediction of primer specificity for eDNA surveillance

Environmental DNA (eDNA) as a tool for assessing fish biomass: A review of approaches and future considerations for resource surveys

Sturgeons in large rivers: detecting the near-extinct needles in a haystack via eDNA metabarcoding from water samples

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