The purpose of this study was to compare low-frequency, rare variant allele detection by digital PCR in Streck Cell-Free DNA BCT samples processed by the NEAT Liquid Biopsy Kit with competitor Kit 2. The data demonstrates that the NEAT Liquid Biopsy Kit increases the concentration of mutant alleles in these DNA samples, which improves the reliability of detection across replicates. In other words, the NEAT Liquid Biopsy Kit has fewer non-detects than competitor Kit 2. This data highlights the performance of the NEAT Liquid Biopsy Kit compared to its competitor, Kit 2, in the detection of rare variant alleles in difficult samples for cfDNA extraction. TECHNICAL NOTE - SKU 10XXX SKU 55XXX

This video provides an overview of the NEAT Liquid Biopsy Kit. Length: 10:01min/sec

Evaluation of an affinity capture method and microfluidic digital PCR as a rapid approach to quantify SARS-CoV-2, mpox virus, and fecal indicator, pepper mild mottle virus (PMMoV) in wastewater during a mass-gathering event Science of The Total Environment, October 2023 Abstract: Wastewater surveillance has emerged recently as a powerful approach to understanding infectious disease dynamics in densely populated zones. Wastewater surveillance, while promising as a public health tool, is often hampered by slow turn-around times, complex analytical protocols, and resource-intensive techniques. In this study, we evaluated an affinity capture method and microfluidic digital PCR as a rapid approach to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mpox (formerly known as monkeypox) virus, and fecal indicator, pepper mild mottle virus (PMMoV) in wastewater during a mass-gathering event. Wastewater samples (n = 131) were collected from residential and commercial manholes, pump stations, and a city's wastewater treatment plant. The use of Nanotrap® Microbiome Particles and microfluidic digital PCR produced comparable results to other established methodologies, with reduced process complexity and analytical times, providing same day results for public health preparedness and response. Using indigenous SARS-CoV-2 and PMMoV in wastewater, the average viral recovery efficiency was estimated at 10.1 %. Both SARS-CoV-2 N1 and N2 genes were consistently detected throughout the sampling period, with increased RNA concentrations mainly in wastewater samples collected from commercial area after festival mass gatherings. The mpox virus was sporadically detected in wastewater samples during the surveillance period, without distinct temporal trends. SARS-CoV-2 RNA concentrations in the city's wastewater mirrored the city's COVID-19 cases, confirming the predictive properties of wastewater surveillance. Wastewater surveillance continues to be beneficial for tracking diseases that display gastrointestinal symptoms, including SARS-CoV-2, and can be a powerful tool for sentinel surveillance. However, careful site selection and a thorough understanding of community dynamics are necessary when performing targeted surveillance during temporary mass-gathering events as potential confirmation bias may occur.

Improved methodology for detection of antibiotic resistance mechanisms from wastewater for population surveillance APPLICATION NOTE SKU 10XXX SKU 44XXX Download full application note here Introduction Antibiotic resistant bacteria, which harbor antimicrobial resistance (AMR) genes, pose a significant public health threat. For these bacteria, traditional means of antibiotic treatment are no longer effective. As such, it is imperative to monitor the prevalence of these bacteria. Recently, wastewater surveillance has been recognized as a complement to population testing that can identify increases in the presence of AMR genes in communities. However, the use of molecular testing methods is challenging because many genetic mechanisms contribute to AMR, with the most common being β-Lactamases. To be an effective surveillance strategy, these tests must be designed to identify a broad range of targets associated with AMR, and the methods must be sensitive given the larger wastewater sampling volumes used for analysis. The data described here demonstrate methods for improved sample processing and a head-to-head comparison of the ability of commercially available real-time PCR-based assays to determine AMR burden in wastewater samples.

Exploring the Potential of Nanotrap® Microbiome Particles and digital PCR for High-Throughput Microbial Source Tracking in Surface Waters Poster: In coastal Louisiana, high water tables and poor soil conditions often require the use of aerobic treatment units (ATUs) for onsite sewage treatment. Owing to ATU treatment failures, coastal surface waters are subject to increased fecal contamination via domestic wastewater discharge. While useful to measure fecal contamination, traditional culture-based bacteriological testing cannot indicate specific sources of contamination for remediation. Microbial source tracking assays, on the other hand, are source-specific molecular tests that can be used to more precisely determine the source of fecal contamination for the targeted response. Standard methods for MST require the use of membrane filtration to concentrate samples for testing, which greatly limits throughput. Nanotrap Microbiome Particles (NMPs) utilize the Microbiome A&B particles’ high affinity for bacteria to attract, capture and enrich low-abundance bacteria in the solution (Ahmed et al., 2023). NMPs can be used in conjunction with liquid handling platforms, such as the KingFisher Apex to greatly increase sample throughput and decrease time to results. But the performance of NMPs for Microbial Source Tracking (MST) in comparison with other concentration workflows has not been evaluated.

Nature Medicine, August 2023 "Environmental surveillance, defined as the systematic collection of samples and associated data on infectious-disease-related pathogens from wastewater for the purpose of informing decisions, has a rich tradition in public health. High-resource settings such as the USA and Europe have started to implement environmental surveillance networks for the use of multi-pathogen data from wastewater, including for pandemic preparedness. Implementing environmental surveillance in lower-resource settings, in which a large proportion of populations live in houses not connected to convergent sewer systems, has lagged due to epidemiological and resource challenges. Correcting this imbalance is important to ensure equitable access to actionable surveillance."

Poster: Wastewater-based epidemiology has been widely used to monitor COVID-19 trends in communities, the spread of SARS-CoV-2 variants in the population, and the effectiveness of public health prevention measures. Since the beginning of the pandemic, there have been five lineages classified as variants of concerns (VOCs) of SASR-CoV-2 (Alpha, Beta, Gamma, Delta, and Omicron) described by the WHO. Since November 2021, significant changes in circulating strains have been primarily Omicron sub-lineages. Variants have been successfully identified and quantified in wastewater using next-generation sequencing (NGS) methods that are time-consuming and expensive. We applied allele-specific dPCR and RT-qPCR assays with sequence-specific primers and fluorescently-labeled oligonucleotide probes developed for genotyping clinical samples to estimate the proportion of four Omicron sub-lineages (BA1, BA2, BQ1, and XBB) in archived wastewater samples. Samples were collected weekly from the influent of several wastewater treatment facilities, public schools, and a county jail in 2022 and 2023 in Atlanta, GA, USA. The assays were applied in proportionally mixed Omicron sub-lineage standards, which indicated that they were sensitive, specific, and quantitatively-reliable. Omicron sub-lineage detection using the assays was aligned well with the NGS results from the same wastewater samples. Wastewater samples collected between mid-February 2022 and mid-April 2022 showed a rapid displacement of BA1 with BA2, and samples collected between mid-December 2022 and early-March 2023 demonstrated a clear transition from BQ1 to XBB. These sub-lineage displacements detected by PCR assays were concordant with these sub-lineage dynamics in clinical samples reported in the literature. These results suggest that dPCR and RT-qPCR based assays can be used for specific and timely detection and monitoring of SARS-CoV-2 variants in wastewater.

"Characterization and Optimization of Nanotrap® Microbiome Particle methods for Concentrating SARS-CoV-2 from Small Volume Wastewater Samples" Frontiers Microbiology, July 2023 Introduction: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA monitoring in wastewater has become an important tool for Coronavirus Disease 2019 (COVID-19) surveillance. Grab (quantitative) and passive samples (qualitative) are two distinct wastewater sampling methods. Although many viral concentration methods such as the usage of membrane filtration and skim milk are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. Methods: The objectives of this study were to compare two workflows (Nanotrap® Microbiome A Particles coupled with MagMax kit and membrane filtration workflows coupled with RNeasy kit) for SARS-CoV-2 recovery in grab samples and two workflows (Nanotrap® Microbiome A Particles and skim milk workflows coupled with MagMax kit) for SARS-CoV-2 recovery in Moore swab samples. The Nanotrap particle workflow was initially evaluated with and without the addition of the enhancement reagent 1 (ER1) in 10 mL wastewater. RT-qPCR targeting the nucleocapsid protein was used for detecting SARS-CoV-2 RNA. Results: Adding ER1 to wastewater prior to viral concentration significantly improved viral concentration results (P < 0.0001) in 10 mL grab and swab samples processed by automated or manual Nanotrap workflows. SARS-CoV-2 concentrations in 10 mL grab and Moore swab samples with ER1 processed by the automated workflow as a whole showed significantly higher (P < 0.001) results than 150 mL grab samples using the membrane filtration workflow and 250 mL swab samples using the skim milk workflow, respectively. Spiking known genome copies (GC) of inactivated SARS-CoV-2 into 10 mL wastewater indicated that the limit of detection of the automated Nanotrap workflow was ~11.5 GC/mL using the RT-qPCR and 115 GC/mL using the digital PCR methods. Discussion: These results suggest that Nanotrap workflows could substitute the traditional membrane filtration and skim milk workflows for viral concentration without compromising the assay sensitivity. The manual workflow can be used in resource-limited areas, and the automated workflow is appropriate for large-scale COVID-19 wastewater-based surveillance.

Science of The Total Environment, June 2023 "Unveiling indicator, enteric, and respiratory viruses in aircraft lavatory wastewater using adsorption-extraction and Nanotrap® Microbiome A Particles workflows" Abstract: The effective detection of viruses in aircraft wastewater is crucial to establish surveillance programs for monitoring virus spread via aircraft passengers. This study aimed to compare the performance of two virus concentration workflows, adsorption-extraction (AE) and Nanotrap® Microbiome A Particles (NMAP), in detecting the prevalence and concentrations of 15 endogenous viruses including ssDNA, dsDNA, ssRNA in 24 aircraft lavatory wastewater samples. The viruses tested include two indicator viruses, four enteric viruses, and nine respiratory viruses. The results showed that cross-assembly phage (crAssphage), human polyomavirus (HPyV), rhinovirus A (RhV A), and rhinovirus B (RhV B) were detected in all wastewater samples using both workflows. However, enterovirus (EV), human norovirus (HNoV GII), human adenovirus (HAdV), bocavirus (BoV), parechovirus (PeV), epstein-barr virus (EBV), influenza A virus (IAV), and respiratory syncytial virus B (RsV B) were infrequently detected by both workflows, and hepatitis A virus (HAV), influenza B virus (IBV), and respiratory syncytial virus B (RsV A) were not detected in any samples. The NMAP workflow had greater detection rates of RNA viruses (EV, PeV, and RsV B) than the AE workflow, while the AE workflow had greater detection rates of DNA viruses (HAdV, BoV, and EBV) than the NMAP workflow. The concentration of each virus was also analyzed, and the results showed that crAssphage had the highest mean concentration (6.76 log10 GC/12.5 mL) followed by HPyV (5.46 log10 GC/12.5 mL using the AE workflow, while the mean concentrations of enteric and respiratory viruses ranged from 2.48 to 3.63 log10 GC/12.5 mL. Using the NMAP workflow, the mean concentration of crAssphage was 5.18 log10 GC/12.5 mL and the mean concentration of HPyV was 4.20 log10 GC/12.5 mL, while mean concentrations of enteric and respiratory viruses ranged from 2.55 to 3.74 log10 GC/12.5 mL. Significantly higher (p < 0.05) mean concentrations of crAssphage and HPyV were observed when employing the AE workflow in comparison to the NMAP workflow. Conversely, the NMAP workflow yielded significantly greater (p < 0.05) concentrations of RhV A, and RhV B compared to the AE workflow. The findings of this study can aid in the selection of an appropriate concentration workflow for virus surveillance studies and contribute to the development of efficient and reliable virus detection methods.

"Intensity of sample processing methods impacts wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes" Science of The Total Environment, June 2023 ABSTRACT: Wastewater SARS-CoV-2 surveillance has been deployed since the beginning of the COVID-19 pandemic to monitor dynamics in virus burden in local communities. Genomic surveillance of SARS-CoV-2 in wastewater, particularly the efforts for whole genome sequencing for variant tracking or identification, are comparatively challenging due to low target concentration, complex microbial and chemical background, and lack of robust nucleic acid recovery experimental procedures. The intrinsic sample limitations are inherent to wastewater. In this study, we evaluated impacts from sample types, certain sample intrinsic features, and processing and sequencing methods on sequencing outcomes with a specific focus on the breadth of genome coverage. We collected 184 composite and grab wastewater samples from the Chicago area between March to October 2021 for SARS-CoV-2 quantification and genomic surveillance. Samples were processed using a mixture of processing methods reflecting different homogenization intensities (HA+Zymo beads, HA+glass beads, and Nanotrap), and were sequenced using two sequencing library preparation kits (the Illumina COVIDseq kit and the QIAseq DIRECT kit). A synthetic SARS-CoV-2 RNA experiment was performed to validate the potential impacts of processing methods on sequencing outcomes. Our findings suggested that 1) wastewater SARS-CoV-2 whole genome sequencing outcomes were associated with sample types and processing methods 2) in less intensive method processed samples (HA+glass beads), higher genome breadth of coverage in sequencing (over 80%) was associated with N1 concentration > 105 cp/L, while in intensive method (HA+Zymo beads), qPCR results were inconsistent with sequencing outcomes, and 3) sample processing methods and sequencing kits, rather than the extraction methods or intrinsic features of wastewater samples, played important roles in wastewater SARS-CoV-2 amplicon sequencing. Overall, extra attention should be paid to wastewater sample processing (e.g., concentration and homogenization) for sufficient, good quality RNA yield for downstream sequencing.

medRxiv May 2023 Background The origin of divergent SARS-CoV-2 spike sequences found in wastewater, but not in clinical surveillance, remains unclear. These “cryptic” wastewater sequences have harbored many of the same mutations that later emerged in Omicron lineages. We first detected a cryptic lineage in municipal wastewater in Wisconsin in January 2022. Named the “Wisconsin Lineage”, we sought to determine this virus’s geographic origin and characterize its persistence and evolution over time. Methods We systematically sampled maintenance holes to trace the Wisconsin Lineage’s origin. We sequenced spike RBD domains, and where possible, whole viral genomes, to characterize the evolution of this lineage over the 13 consecutive months that it was detectable. Findings The persistence of the Wisconsin Lineage signal allowed us to trace it from a central wastewater plant to a single facility, with a high concentration of viral RNA. The viral sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in Wisconsin at low levels from October 2020 to February 2021, while mutations in the spike gene resembled those subsequently found in Omicron variants. Interpretation We propose that prolonged detection of the Wisconsin Lineage in wastewater represents persistent shedding of SARS-CoV-2 from an infected individual, with ongoing within-host viral evolution leading to an ancestral B.1.234 virus accumulating “Omicron-like” mutations.

medRxiv, May 2023 "Human norovirus (HuNoV) GII RNA in wastewater solids at 145 United States wastewater treatment plants: Comparison to positivity rates of clinical specimens and modeled estimates of HuNoV GII shedders" Background: Human norovirus (HuNoV) is a leading cause of disease globally, yet actual incidence is unknown. HuNoV infections are not reportable in the United States, and surveillance is limited to tracking severe illnesses or outbreaks. Wastewater monitoring for HuNoV has been done previously and results indicate it is present in wastewater influent and concentrations are associated with HuNoV infections in the communities contributing to wastewater. However, work has mostly been limited to monthly samples of liquid wastewater at one or a few wastewater treatment plants (WWTPs). Methods: We measured HuNoV GII RNA in matched wastewater solids and liquid influent in 7 samples from a WWTP. We also applied the HuNoV GII assay to measure viral RNA in over 6000 wastewater solids samples from 145 WWTPs from across the United States daily to three times per week for up to five months. Measurements were made using digital droplet RT-PCR. Results: HuNoV GII RNA preferentially adsorbs to wastewater solids where it is present at 1000 times the concentration in influent. Concentrations of HuNoV GII RNA correlate positively with clinical HuNoV positivity rates. Model output of the fraction of individuals shedding HuNoV is variable and uncertain, but consistent with i Interpretation: These findings support the utility of wastewater solids as a matrix for community infectious disease surveillance.