A cheap,reliable and rapid method of extracting high‐quality DNA from plants

Here we describe a rapid method for extracting DNA from plant material using a microtitre plate system. This extraction procedure has been tested using three species, Brassica napus, Brassica napus/rapa hybrids and Arum maculatum, stored frozen, in silica gel or used fresh. The length of storage was between 7 days and 2 years, and in all cases high molecular weight DNA was reliably purified. Polymerase chain reaction (PCR) testing, using multiplex and single product amplification, inter simple sequence repeats (ISSRs) and microsatellites, was always reliable. This method combines the speed of commercial 96‐well plate methods with the economies associated with readily available laboratory chemicals.     

Evaluating the interaction of faecal pellet deposition rates and DNA degradation rates to optimize sampling design for DNA‐based mark–recapture analysis of Sonoran pronghorn

Knowledge of population demographics is important for species management but can be challenging in low‐density, wide‐ranging species. Population monitoring of the endangered Sonoran pronghorn (Antilocapra americana sonoriensis) is critical for assessing the success of recovery efforts, and noninvasive DNA sampling (NDS) could be more cost‐effective and less intrusive than traditional methods. We evaluated faecal pellet deposition rates and faecal DNA degradation rates to maximize sampling efficiency for DNA‐based mark–recapture analyses. Deposition data were collected at five watering holes using sampling intervals of 1–7 days and averaged one pellet pile per pronghorn per day. To evaluate nuclear DNA (nDNA) degradation, 20 faecal samples were exposed to local environmental conditions and sampled at eight time points from one to 124 days. Average amplification success rates for six nDNA microsatellite loci were 81% for samples on day one, 63% by day seven, 2% by day 14 and 0% by day 60. We evaluated the efficiency of different sampling intervals (1–10 days) by estimating the number of successful samples, success rate of individual identification and laboratory costs per successful sample. Cost per successful sample increased and success and efficiency declined as the sampling interval increased. Results indicate NDS of faecal pellets is a feasible method for individual identification, population estimation and demographic monitoring of Sonoran pronghorn. We recommend collecting samples >7 days old and estimate that a sampling interval of 4–7 days in summer conditions (i.e. extreme heat and exposure to UV light) will achieve desired sample sizes for mark–recapture analysis while also maximizing efficiency.     

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans

Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR‐based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria‐specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro‐organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.     

An inexpensive and high‐throughput procedure to extract and purify total genomic DNA for population studies

We describe here a procedure for the purification of high molecular weight genomic DNA that combines the economies of ‘do‐it‐yourself’, single‐tube protocols with the sample throughput and DNA quality of microplate‐based DNA extraction and purification kits from commercial suppliers. The procedure allows the preparation of genomic DNA of a quality suitable for polymerase chain reaction‐based studies of large populations at around one‐tenth of the cost of commercially available kits. Furthermore, 96 samples can be purified from crude tissue digests in around 30 min and are produced in microtitre plate format to allow efficient downstream processing of samples.     

Detection and quantification of Erysiphe necator DNA in wine grapes and resultant must and juice

Powdery mildew of grapevines is difficult to assess visually at the weighbridge, particularly in large consignments of machine-harvested fruit. To facilitate accurate methods for the detection and quantification of the disease in grape samples obtained from both the vineyard and winery, we developed a DNA probe for the pathogen Erysiphe necator. The E. necator-specific 450 bp DNA fragment pEnA1, targets highly repetitive sequences and was isolated from a partial genomic library. In screening for species specificity, clone pEnA1 was used in slot-blot hybridization and detected E. necator DNA from grapes and resultant must and juice, but not from clarified juice and wine. The detection threshold was approximately 50 pg ofE. necator DNA per 100 ng total DNA of grape sample and was equivalent to 1–5 % of a grape bunch visually affected by powdery mildew. Disease severity, expressed as the percentage of surface area of a bunch with powdery mildew, and E. necator DNA content were highly correlated, r² = 0.955, P < 0.001. The DNA-based hybridization assay has the potential to predict the severity of powdery mildew in grape samples from the vineyard and in must and juice samples at the winery. The DNA sequence of clone pEnA1 was used to design species-specific primers, the results maintaining the same specificity patterns observed in the initial hybridization assays. The PCR-based assay was sensitive enough to detect approximately 1 pg DNA, being equivalent to 1 conidium per sample. This is the first report to date of the detection of all known phenetic groups of E. necator DNA and of the quantification of DNA from grape samples at the winery. Accurate information on the extent of powdery mildew contamination of grape lots would enable wineries to make more informed decisions about the use of fruit and must.     

A simple and sensitive method for detection of Bacillus anthracis by loop‐mediated isothermal amplification

Aims: To develop a rapid and simple system for detection of Bacillus anthracis using a loop‐mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. Methods and Results: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30–40 min under isothermal conditions at 63°C. No cross‐reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3·6 CFU per test. Conclusions: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. Significance and Impact of the Study: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.     

Nuclear internal transcribed spacer‐1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio

The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.     

Using eggshell membranes as a DNA source for population genetic research

In the context of population genetic research, a faster and less invasive method of DNA sampling would allow large-scale assessments of genetic diversity and genetic differentiation with the help of volunteer observers. The aim of this study was to investigate the usefulness of eggshell membranes as a DNA source for population genetic research, by addressing eggshell membrane DNA quality, degeneration and cross-contamination. To this end, a comparison was made with blood-derived DNA samples. We have demonstrated 100% successful DNA extraction from post-hatched Black-tailed Godwit (Limosa limosa) eggshell membranes as well as from blood samples. Using 11 microsatellite loci, DNA amplification success was 99.1% for eggshell membranes and 97.7% for blood samples. Genetic information within eggshell membrane DNA in comparison to blood DNA was not affected (F _ST = −0.01735, P = 0.999) by degeneration or possible cross-contamination. Furthermore, neither degeneration nor cross-contamination was apparent in total genotypic comparison of eggshell membrane DNA and blood sample DNA. Our research clearly illustrates that eggshell membranes can be used for population genetic research.     

A set of polymorphic microsatellite loci for the bay scallop,Argopecten irradians

The method of creating enriched microsatellite libraries can supply an abundant source of microsatellite sequences at a considerably reduced cost. Here we report the development of 15 polymorphic microsatellite loci from the bay scallop, Argopecten irradians, using enrichment protocol. Polymorphism was assessed in a sample of hatchery population (n = 38) revealing three to seven alleles per locus. The expected and observed heterozygosities ranged from 0.198 to 0.813 and from 0.083 to 0.833, respectively. These markers will be useful for genetic variation monitoring and parentage analysis.     

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. aviumintracellulare). Other possible applications of this method are discussed.     

Isolation and characterization of 12 microsatellite loci from cutlassfish (Trichiurus haumela)

The cutlassfish (Trichiurus haumela) is an important commercial fish species. In the present study, we report 12 polymorphic microsatellite DNA markers for cutlassfish. The number of alleles per locus ranged from 2 to 9 in a sample of 26 individuals. Observed and expected heterozygosities per locus varied from 0.2727 to 0.9583 and from 0.4059 to 0.7926, respectively. Two loci (Trha25 and Trha27) showed significant departure from Hardy–Weinberg equilibrium after sequential Bonferroni correction (P < 0.0042). No significant linkage disequilibrium between pairs of loci was found. These microsatellite markers provide powerful tools for investigating genetic population structure, population history and conservation management of cutlassfish. Jin-Zhen Bi and Chang-Wei Shao contributed equally to this work.     

Protoplast transformation of Kluyveromyces marxianus

The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0 °C with carrier DNA, CaCl₂, and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3–4% agar and 0.8 m sorbitol. By using the optimized method, plasmids of 10, 24, and 58 kb were successfully transformed into K. marxianus. The highest efficiency reached 1.8 × 10⁴ transformants per μg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.     

New approaches to species delimitation and population structure of anthozoans: Two case studies of octocorals using ultraconserved elements and exons

As coral populations decline worldwide in the face of ongoing environmental change, documenting their distribution, diversity and conservation status is now more imperative than ever. Accurate delimitation and identification of species is a critical first step. This task, however, is not trivial as morphological variation and slowly evolving molecular markers confound species identification. New approaches to species delimitation in corals are needed to overcome these challenges. Here, we test whether target enrichment of ultraconserved elements (UCEs) and exons can be used for delimiting species boundaries and population structure within species of corals by focusing on two octocoral genera, Alcyonium and Sinularia, as exemplary case studies. We designed an updated bait set (29,181 baits) to target‐capture 3,023 UCE and exon loci, recovering a mean of 1,910 ± 168 SD per sample with a mean length of 1,055 ± 208 bp. Similar numbers of loci were recovered from Sinularia (1,946 ± 227 SD) and Alcyonium (1,863 ± 177 SD). Species‐level phylogenies were highly supported for both genera. Clustering methods based on filtered single nucleotide polymorphisms delimited species and populations that are congruent with previous allozyme, DNA barcoding, reproductive and ecological data for Alcyonium, and offered further evidence of hybridization among species. For Sinularia, results were congruent with those obtained from a previous study using restriction site associated DNA sequencing. Both case studies demonstrate the utility of target‐enrichment of UCEs and exons to address a wide range of evolutionary and taxonomic questions across deep to shallow timescales in corals.     

Transferability of a tetracycline resistance gene from probiotic <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> to bacteria in the gastrointestinal tract of humans

The potential of Lactobacillus reuteri as a donor of antibiotic resistance genes in the human gut was investigated by studying the transferability of the tetracycline resistance gene tet(W) to faecal enterococci, bifidobacteria and lactobacilli. In a double-blind clinical study, seven subjects consumed L. reuteri ATCC 55730 harbouring a plasmid-encoded tet(W) gene (tet(W)-reuteri) and an equal number of subjects consumed L. reuteri DSM 17938 derived from the ATCC 55730 strain by the removal of two plasmids, one of which contained the tet(W) gene. Faecal samples were collected before, during and after ingestion of 5 × 10⁸ CFU of L. reuteri per day for 14 days. Both L. reuteri strains were detectable at similar levels in faeces after 14 days of intake but neither was detected after a two-week wash-out period. After enrichment and isolation of tetracycline resistant enterococci, bifidobacteria and lactobacilli from each faecal sample, DNA was extracted and analysed for presence of tet(W)-reuteri using a real-time PCR allelic discrimination method developed in this study. No tet(W)-reuteri signal was produced from any of the DNA samples and thus gene transfer to enterococci, bifidobacteria and lactobacilli during intestinal passage of the probiotic strain was non-detectable under the conditions tested, although transfer at low frequencies or to the remaining faecal bacterial population cannot be excluded.     

Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit

Noninvasive genetic sampling of faecal pellets can be a valuable method for monitoring rare and cryptic wildlife populations, like the pygmy rabbit (Brachylagus idahoensis). To investigate this method's efficiency for pygmy rabbit monitoring, we evaluated the effect of sample age on DNA degradation in faecal pellets under summer field conditions. We placed 275 samples from known individuals in natural field conditions for 1–60 days and assessed DNA quality by amplifying a 294‐base‐pair (bp) mitochondrial DNA (mtDNA) locus and five nuclear DNA (nDNA) microsatellite loci (111–221 bp). DNA degradation was influenced by sample age, DNA type, locus length and rabbit sex. Both mtDNA and nDNA exhibited high PCR success rates (94.4%) in samples <1 day old. Success rates for microsatellite loci declined rapidly from 80.0% to 42.7% between days 5 and 7, likely due to increased environmental temperature. Success rates for mtDNA amplification remained higher than nDNA over time, with moderate success (66.7%) at 21 days. Allelic dropout rates were relatively high (17.6% at <1 day) and increased to 100% at 60 days. False allele rates ranged from 0 to 30.0% and increased gradually over time. We recommend collecting samples as fresh as possible for individual identification during summer field conditions. Our study suggests that this method can be useful for future monitoring efforts, including occupancy surveys, individual identification, population estimation, parentage analysis and monitoring of genetic diversity both of a re‐introduced population in central Washington and across their range.     

Compared with conventional PCR assay,qPCR assay greatly improves the detection efficiency of predation

Studies of predation can contribute greatly to understanding predator–prey relationships and can also provide integral knowledge concerning food webs and multi‐trophic level interactions. Both conventional polymerase chain reaction (cPCR) and quantitative PCR (qPCR) have been employed to detect predation in the field because of their sensitivity and reproducibility. However, to date, few studies have been used to comprehensively demonstrate which method is more sensitive and reproducible in studies of predation. We used a Drosophila melanogaster‐specific DNA fragment (99 bp) to construct a tenfold gradient dilution of standards. Additionally, we obtained DNA samples from Pardosa pseudoannulata individuals that fed on D. melanogaster at various time since feeding. Finally, we compared the sensitivity and reproducibility between cPCR and qPCR assays for detecting DNA samples from feeding trials and standards. The results showed that the cPCR and qPCR assays could detect as few as 1.62 × 10³ and 1.62 × 10¹ copies of the target DNA fragment, respectively. The cPCR assay could detect as few as 48 hr post‐feeding of the target DNA fragment. But the qPCR assay showed that all spiders were positive after consuming prey at various time intervals (0, 24, 48, 72, and 96 hr). A smaller proportion of the technical replicates were positive using cPCR, and some bands on the agarose gel were absent or gray, while some were white and bright for the same DNA samples after amplification by cPCR. By contrast, a larger proportion of the technical replicates were positive using qPCR and the coefficients of variation of the Ct value for the three technical replicates of each DNA sample were less than 5%. These data showed that qPCR was more sensitive and highly reproducible in detecting such degraded DNA from predator's gut. The present study provides an example of the use of cPCR and qPCR to detect the target DNA fragment of prey remains in predator's gut.     

New SNPs for population genetic analysis reveal possible cryptic speciation of eastern Australian sea mullet (Mugil cephalus)

Sustainable management of sea mullet (Mugil cephalus) fisheries needs to account for recent observations of regional‐scale differentiation. Population genetic analysis is sought to assess the situation of this ecologically and economically important fish species in eastern Australian waters. Here, we report (i) new population genetic markers [single nucleotide polymorphisms (SNPs) and potential microsatellites], (ii) first estimates of spatial genetic differentiation and (iii) prospective power tests for designing more comprehensive studies. Six DNA samples from three sampling regions (North Queensland, South Queensland and central New South Wales) on the eastern coast of Australia were used to prepare restriction site associated DNA (RAD) tag libraries from genomic DNA digested with EcoRI and MseI. A pooled sample of regional RAD tag libraries was sequenced using the Roche GS‐FLX Titanium platform. A total of 172 837 raw reads (17.4 Mbp) were retrieved, 95 500 of which were used to discover 1267 SNPs and 1417 microsatellites. A subset of 161 SNPs was validated based on 63 additional DNA samples genotyped using the Sequenom MassArray (iPLEX Gold chemistry). Altogether 92 SNPs (57%) were confirmed, with 40% of these marking fixed variants between northern and southern sampling regions. Our preliminary findings indicate a multispecies fishery stock of M. cephalus in eastern Australian waters, but suggest that strong genetic differentiation occurs north of major fishing grounds. Low potential differentiation within major fishing grounds (e.g. F_ST = 0.0025) can be resolved with a likely power ≥67% by using standard sample sizes of 50 and validated subsets of available markers.     

Detection of the endangered European weather loach (Misgurnus fossilis) via water and sediment samples: Testing multiple eDNA workflows

The European weather loach (Misgurnus fossilis) is classified as highly endangered in several countries of Central Europe. Populations of M. fossilis are predominantly found in ditches with low water levels and thick sludge layers and are thus hard to detect using conventional fishing methods. Therefore, environmental DNA (eDNA) monitoring appears particularly relevant for this species. In previous studies, M. fossilis was surveyed following eDNA water sampling protocols, which were not optimized for this species. Therefore, we created two full factorial study designs to test six different eDNA workflows for sediment samples and twelve different workflows for water samples. We used qPCR to compare the threshold cycle (C_t) values of the different workflows, which indicate the target DNA amount in the sample, and spectrophotometry to quantify and compare the total DNA amount inside the samples. We analyzed 96 water samples and 48 sediment samples from a pond with a known population of M. fossilis. We tested several method combinations for long‐term sample preservation, DNA capture, and DNA extraction. Additionally, we analyzed the DNA yield of samples from a ditch with a natural M. fossilis population monthly over one year to determine the optimal sampling period. Our results showed that the long‐term water preservation method commonly used for eDNA surveys of M. fossilis did not lead to optimal DNA yields, and we present a valid long‐term sample preservation alternative. A cost‐efficient high salt DNA extraction led to the highest target DNA yields and can be used for sediment and water samples. Furthermore, we were able to show that in a natural habitat of M. fossilis, total and target eDNA were higher between June and September, which implies that this period is favorable for eDNA sampling. Our results will help to improve the reliability of future eDNA surveys of M. fossilis.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

Use of environmental DNA to detect Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in large Laurentian Great Lakes embayments

A single-species environmental DNA (eDNA) method was developed to sample for a small, benthic rare species, Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in two large Lake Ontario embayments. Summer water sampling allowed for: (a) surveys of habitats (Wellers Bay) where traditional fish sampling gear could not be used; and, (b) a comparison between eDNA and seining-based detection probabilities at known occupied habitats (West Lake). In 2018, replicate (n = 3) 1 L water samples were collected from 90 Wellers Bay sites and 71 West Lake sites. A site-occupancy model, a hierarchical logistic regression model, was fitted to determine site occupancy, sample occupancy (presence of Eastern Sand Darter DNA in a water sample) and probability of detection (p) based on replicate quantitative polymerase chain reaction (qPCR) results for each water sample. Eastern Sand Darter was detected at 10 West Lake sites, but not from Wellers Bay. Mean site occupancy was 0.31 (0.12–0.70; 95% CLs), mean sample occupancy was 0.28 (0.09–0.58; 95% CLs), and mean detection probability in a subsample (i.e., successful qPCR amplification) given it was present was 0.40 (0.25–0.55; 95% CLs). While the eDNA method successfully detected Eastern Sand Darter from known occupied areas in West Lake, it was not more effective for assessing local site occupancy than traditional sampling methods, such as the seine.