Development and validation of rapid environmental DNA (eDNA) detection methods for bog turtle (Glyptemys muhlenbergii)

Abstract

Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle’s wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, most often wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Here, we describe the development and validation of a qPCR assay, BT3, targeting the mitochondrial cytochrome oxidase I gene that correctly identifies bog turtles with 100% specificity and sensitivity when tested on 201 blood samples collected from six species over a wide geographic range. We also developed a full-process internal control employing a genetically modified strain of Caenorhabditis elegans to improve DNA extraction methods, limit false negative results due to qPCR inhibition, and measure total DNA recovery from each sample. Using the internal control, we found that DNA recovery varied by over an order of magnitude between samples and likely explains the lack of bog turtle detection in some cases. Methods presented herein are highly-specific and may offer a more cost effective, non-invasive tool to supplement bog turtle population assessments in the Eastern United States. Poor or differential DNA recovery, which remains unmeasured in the vast majority of eDNA studies, significantly reduced the ability to detect bog turtle in their natural environment.

Co-quantification of crAssphage increases confidence in wastewater-based epidemiology for SARS-CoV-2 in low prevalence areas

“…In addition to the research needs relevant to particle binding and decay, as well as the continued methodological and processing refinements mentioned previously, SARS-CoV-2 strain identification from wastewater should be a priority of ongoing WBE for COVID-19. While sequencing approaches have been used successfully to detect variants from wastewater ( Crits-Christoph et al., 2021 ; Jahn et al., 2021 , Kirtane et al., 2019 ), these methods are relatively low throughput. The rapid development of standardized PCR-based assays to detect variants from wastewater would allow sensitive and early detection of variants that pose elevated health risks.…”

“…Kinetic outlier detection (KOD) was performed as described previously ( Green and Field, 2012 ; Kirtane et al., 2019 ; Tichopad et al., 2010 ) on all 3,032 reactions to determine if qPCR inhibition affected the amplification of SARS-CoV-2 or crAssphage nucleic acid targets. Raw fluorescence data from each well were log-transformed and fit to a 4-parameter sigmoidal model using the pcrbatch function in R package qpcR version 1.4-1 ( Ritz and Spiess, 2008 ; Spiess, 2018 ).…”

Landscape, Water Quality, and Weather Factors Associated With an Increased Likelihood of Foodborne Pathogen Contamination of New York Streams Used to Source Water for Produce Production

“…These filters were then used for detection of FST markers specific to avian, canine, human and ruminant sources as previously described (Table 1). Filters were allowed to thaw prior to adding 29.2 µl of prepared Caenorhabditis elegans lysate (Kirtane et al, 2019). The C. elegans strain used here contains a gfp gene, which can be targeted using the CG4 assay, allowing for (i) estimation of the total amount of DNA recovered from each sample, and (ii) confirmation that qPCR inhibition was absent (Kirtane et al, 2019).…”

“…Filters were allowed to thaw prior to adding 29.2 µl of prepared Caenorhabditis elegans lysate (Kirtane et al, 2019). The C. elegans strain used here contains a gfp gene, which can be targeted using the CG4 assay, allowing for (i) estimation of the total amount of DNA recovered from each sample, and (ii) confirmation that qPCR inhibition was absent (Kirtane et al, 2019). Following the addition of the C. elegans lysate, the filter and lysate were homogenized using a FastPrep-24-5G (Irvine, CA, MPBio).…”

Integrative Survey of 68 Non-overlapping Upstate New York Watersheds Reveals Stream Features Associated With Aquatic Fecal Contamination

“…Filters were transferred to Lysing Matrix E tubes (MPBio, Irvine, CA, United States) and stored at −80 • C for between 55 and 218 days 1 newa.cornell.edu (mean = 197 days) until DNA extraction. Prior to extraction, filters were allowed to thaw to room temperature, and a 29.2 µl aliquot of prepared Caenorhabditis elegans lysate was added to each tube as described previously (Kirtane et al, 2019). Following thawing and lysate addition, filters were homogenized on a FastPrep-24-5G (MPBio), and DNA was extracted with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) with a final elution volume of 100 µl.…”