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A minimally invasive, field-applicable CRISPR/Cas biosensor to aid in the detection of Pseudogymnoascus destructans, the causative fungal agent of white-nose syndrome in bats

The accessibility to CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) genetic tools has given rise to applications beyond site-directed genome editing for the detection of DNA and RNA. These tools include precise diagnostic detection of human disease pathogens, such as SARS-CoV-2 and Zika virus. Despite the technology being rapid and cost-effective, the use of CRISPR/Cas tools in the surveillance of the causative agents of wildlife diseases has not been prominent. This study presents the development of a minimally invasive, field-applicable and user-friendly CRISPR/Cas-based biosensor for the detection of Pseudogymnoascus destructans ( Pd ), the causative fungal agent of white-nose syndrome (WNS), an infectious disease that has killed more than five million bats in North America since its discovery in 2006. The biosensor assay combines a recombinase polymerase amplification (RPA) step followed by CRISPR/Cas12a nuclease cleavage to detect Pd DNA from bat dermal swab and guano samples. The biosensor had similar detection results when compared to quantitative PCR in distinguishing Pd- positive versus negative field samples. Although bat dermal swabs could be analysed with the biosensor without nucleic acid extraction, DNA extraction was needed when screening guano samples to overcome inhibitors. This assay can be applied to help with more rapid delineation of Pd- positive sites in the field to inform management decisions. With further optimization, this technology has broad translation potential to wildlife disease-associated pathogen detection and monitoring applications.

Molecular Ecology Resources

Early detection of wildlife disease pathogens using CRISPR-Cas system methods

Wildlife diseases are a considerable threat to human health, conservation, and the economy. Surveillance is a critical component to mitigate the impact of animal diseases in these sectors. To monitor human diseases, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) biosensors have proven instrumental as diagnostic tools capable of detecting unique DNA and RNA sequences related to their associated pathogens. However, despite the significant advances in the general development of CRISPR-Cas biosensors, their use to support wildlife disease management is lagging. In some cases, wildlife diseases of concern could be rapidly surveyed using these tools with minimal technical, operational, or cost requirements to end users. This review explores the potential to further leverage this technology to advance wildlife disease monitoring and highlights how concerted standardization of protocols can help to ensure data reliability.

The CRISPR Journal

A brief note on substantial sub-daily arsenic variability in pumping drinking-water wells in New Hampshire

Large variations in redox-related water parameters, like pH and dissolved oxygen (DO), have been documented in New Hampshire (United States) drinking-water wells over the course of a few hours under pumping conditions. These findings suggest that comparable sub-daily variability in dissolved concentrations of redox-reactive and toxic arsenic (As) also may occur, representing a potentially critical public-health data gap and a fundamental challenge for long-term As-trends monitoring. To test this hypothesis, discrete groundwater As samples were collected approximately hourly during one day in May and again in August 2019 from three New Hampshire drinking-water wells (2 public-supply, 1 private) under active pumping conditions. Collected samples were assessed by laboratory analysis (total As [As Tot ], As(III), As(V)) and by field analysis (As Tot ) using a novel integrated biosensor system. Laboratory analysis revealed sub-daily variability (range) in As Tot concentrations equivalent to 16 % – 36 % of that observed in the antecedent 3-year bimonthly trend monitoring. Thus, the results indicated that, along with previously demonstrated seasonality effects, the timing and duration of pumping are important considerations when assessing trends in drinking-water As exposures and concomitant risks. Results also illustrated the utility of the field sensor for monitoring and management of As Tot exposures in near-real-time.

Science of the Total Environment

Microbial and viral indicators of pathogens and human health risks from recreational exposure to waters impaired by fecal contamination

Fecal indicator bacteria (FIB) (e.g., fecal coliforms, Escherichia coli , and enterococci) have been used for decades to monitor for and protect the public from waterborne pathogens from fecal contamination. However, FIB may not perform well at predicting the presence of waterborne pathogens or human health outcomes from recreational exposure to fecal-contaminated surface waters. Numerous factors can influence the relationship between FIB and pathogens or human health outcomes, including the source(s) of contamination, the type of pathogen(s) present, differences in the survival and behavior of FIB and pathogens in the wastewater conveyance and treatment process, and varying environmental conditions. As a result, different indicators, such as source-specific microbial source tracking (MST) markers and viral fecal indicators, have been used as possible surrogates to better approximate pathogen abundance and human health risks in recreational waters. The performance of these alternative indicators has been mixed, with some promise of viral indicators better approximating viral pathogens than bacterial fecal indicators, and FIB generally more closely associated with bacterial and protozoal pathogen presence than human MST markers. Many of the assays to detect and quantify fecal indicators and pathogens are polymerase chain reaction-based assays, which detect and quantify nucleic acid [deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)] sequences specific to a target of interest. Recent advances in DNA and RNA sequencing technologies may push the field toward metabarcoding approaches, where multiple targets can be detected and quantified simultaneously. Metabarcoding is currently more applicable to bacterial and protozoal assessments than viral assessments based on a lack of universal metabarcoding markers for viruses. Innovative technologies, such as biosensors and nanotechnologies, may provide more sensitive and accurate tools to detect and quantify pathogens. When a specific pathogen is of concern for a recreational water body, a practical approach in estimating the likelihood of human health outcomes is the application of quantitative microbial risk assessments (QMRAs). Quantitative microbial risk assessments can be used to model the likelihood of pathogen-specific human health outcomes from recreational exposure as a function of a surrogate indicator. Inputs for QMRAs include the ratio between the indicator to be monitored and the pathogen of interest, the concentration of the indicator, the amount of water ingested, and the likelihood of the health outcome based on the estimated amount of pathogen consumed. There are numerous unknowns about the behavior and survival of fecal indicators and pathogens in environmental waters. Developing accurate models to predict pathogen concentrations from fecal indicators in recreational waters will require a better understanding of these unknowns. Current methods and technologies for detecting and quantifying fecal indicators and pathogens are limited due to the rare and patchy nature of pathogens. Technological advances may help improve sensitivity for detecting and quantifying pathogens.

Journal of Sustainable Water in the Built Environm

Development of high surface area organosilicate nanoparticulate thin films for use in sensing hydrophobic compounds in sediment and water

The scope of this study was to apply advances in materials science, specifically the use of organosilicate nanoparticles as a high surface area platform for passive sampling of chemicals or pre-concentration for active sensing in multiple-phase complex environmental media. We have developed a novel nanoporous organosilicate (NPO) film as an extraction phase and proof of concept for application in adsorbing hydrophobic compounds in water and sediment. We characterized the NPO film properties and provided optimization for synthesis and coatings in order to apply the technology in environmental media. NPO films in this study had a very high surface area, up to 1325 m 2 /g due to the high level of mesoporosity in the film. The potential application of the NPO film as a sorbent phase for sensors or passive samplers was evaluated using a model hydrophobic chemical, polychlorinated biphenyls (PCB), in water and sediment. Sorption of PCB to this porous high surface area nanoparticle platform was highly correlated with the bioavailable fraction of PCB measured using whole sediment chemistry, porewater chemistry determined by solid-phase microextraction fiber methods, and the Lumbriculus variegatus bioaccumulation bioassay. The surface-modified NPO films in this study were found to highly sorb chemicals with a log octanol-water partition coefficient (K ow ) greater than four; however, surface modification of these particles would be required for application to other chemicals.

Biosensors