APPLICATION NOTE SKU 44XXX Download full application note here MACHERY NAGEL: SARS-CoV-2 extraction from wastewater concentrated with Nanotrap® Magnetic Virus Particles Automatable concentration and purification of viral RNA from wastewater Introduction Many researchers, private companies, and public health agencies are utilizing wastewater testing to monitor COVID-19 infections in selected communities, campus dormitories, or health centers. Wastewater surveillance efforts can be used to look for early warning signs of an upcoming rise in infection rates, and the methods employed by these researchers can be used to estimate numbers of infected individuals who may or may not be symptomatic. Routine wastewater surveillance may prove to be an efficient, non-invasive tool to detect not only COVID-19 infections due to the SARS-CoV-2 virus, but other types of future viral outbreaks as well. Viral RNA extraction from wastewater is a major challenge for laboratories due to the presence of so many substances that cause inhibition of downstream analysis, and because large volumes of wastewater are often needed for detecting the presence of the SARS-CoV-2 virus. Commonly used wastewater concentration methods tend to exacerbate the problem since they also usually concentrate the inhibitors as well. Many concentration techniques are also labor intensive and costly. In this application note we show an efficient, cost-effective, and even automatable method to extract SARS-CoV-2 viral RNA from complex wastewater samples by pairing MACHEREY‑NAGEL’s NucleoMag® DNA/RNA Water kit for extraction with the Nanotrap® Magnetic Virus Particles from Ceres Nanosciences for wastewater concentration

APPLICATION NOTE SKU 10XXX SKU 44XXX Download full application note here Key Advantages >> Nanotrap® Enhancement Reagent 1 improves SARS-CoV-2 RNA detection in wastewater by at least 1-2 Ct values. >> Compatible with RNA extraction kits from multiple vendors. >> Automated methods do not add time to workflow. >> Outperforms other magnetic virus binding beads and standard concentration methods. Introduction According to the Centers for Disease Control and Prevention, SARS-CoV-2 can be shed in the feces of individuals with asymptomatic or symptomatic infections, making SARS-CoV-2 RNA detection in sewage a COVID-19 indicator that is independent of healthcare-seeking behaviors and access to clinical testing. Due to low viral concentrations and presence of PCR inhibitors in sewage samples, SARS-CoV-2 viral particles must be concentrated prior to nucleic acid isolation. Common approaches for viral particle concentration include HA filtration, skim milk and PEG precipitation methods. These approaches are time consuming and cumbersome. Nanotrap® Magnetic Virus particles have already been demonstrated to enable sensitive, rapid, and easy-to-use automated and manual methods for wastewater SARS-CoV-2 testing. A new reagent from Ceres Nanosciences, Nanotrap® Enhancement Reagent 1 (ER1), improves the binding of viruses to Nanotrap® Magnetic Virus Particles in wastewater samples, thus improving downstream detection of viral RNA. This simple and sensitive method is compatible with magnetic bead RNA extraction kits from MACHERY-NAGEL and ThermoFisher.

American Society For Microbiology, mSystems, July 2021 Rapid, large-scale wastewater surveillance and automated reporting system enabled early detection of nearly 85% of COVID-19 cases on a University campus ABSTRACT: Wastewater based surveillance has gained prominence and come to the forefront as a leading indicator of forecasting COVID-19 infection dynamics owing to its cost-effectiveness and its ability to inform early public health interventions. A university campus could especially benefit from wastewater surveillance as they are characterized by largely asymptomatic populations and are potential hotspots for transmission that necessitate frequent diagnostic testing. In this study, we employed a large-scale GIS (Geographic information systems) enabled building-level wastewater monitoring system associated with the on-campus residences of 7614 individuals. Sixty-eight automated wastewater samplers were deployed to monitor 239 campus buildings with a focus on residential buildings. Time-weighted composite samples were collected on a daily basis and analyzed within the same day. Sample processing was streamlined significantly through automation, reducing the turnaround time by 20-fold and exceeding the scale of similar surveillance programs by 10 to 100-fold, thereby overcoming one of the biggest bottlenecks in wastewater surveillance. An automated wastewater notification system was developed to alert residents to a positive wastewater sample associated with their residence and to encourage uptake of campus-provided asymptomatic testing at no charge. This system, integrated with the rest of the 'Return to Learn' program at UC San Diego-led to the early diagnosis of nearly 85% of all COVID-19 cases on campus. Covid-19 testing rates increased by 1.9-13X following wastewater notifications. Our study shows the potential for a robust, efficient wastewater surveillance system to greatly reduce infection risk as college campuses and other high-risk environments reopen.

POSTER Presented at 2020 AMP Annual Meeting & Expo, November 2020 Introduction: Liquid biopsies, especially those that use plasma ctDNA, are emerging as a powerful complement, and, in some cases, alternative to solid tumor biopsies for the molecular characterization of cancer. Nonsmall cell lung cancer (NSCLC) has proven particularly amenable to liquid biopsies due to the availability of an FDA-approved tyrosine kinase inhibitor for patients whose tumors have EGFR exon 19 deletions or exon 21 L858R mutations, as well as the need to monitor disease progression and treatment response. However, collection of ctDNA is challenged by the low abundance of ctDNA in blood, the potential for contamination by genomic DNA, as well as other factors. These challenges can lead to false negative results that limit utility of liquid biopsy. Methods: We compared the recovery of ctDNA using a commercial column-based approach (Roche) to a novel magnetic hydrogel particle (Ceres Nanosciences) method. We created contrived liquid biopsy specimens by spiking fragmented EGFR wild-type and mutant DNA sequences into pooled donor plasma at varying concentrations. Recovered DNA was quantitated and tested for EGFR mutations using a semi-quantitative multiplex PCR assay (COBAS 4800). The performance of these 2 ctDNA extraction methods were further compared using plasma derived from NSCLC patients with known mutational profiles. Results: In the contrived specimens, the column-based approach recovered 48% to 63% of spiked DNA, whereas the magnetic hydrogel particles recovered 79% to 84% of spiked DNA. Importantly, the hydrogel particle-based approach prevented genomic DNA contamination for up to 24 hours post-collection. Extracted ctDNA from both approaches demonstrated comparable performance in the detection of EGFR mutations, with EGFR exon19del, L858R, and T790M mutation detection possible at plasma DNA concentrations of 0.5, 4, and 20 ng/mL, respectively. Patient data demonstrated concordance between ctDNA and tissue analysis in most cases, regardless of ctDNA extraction method. ctDNA analysis revealed an EGFR L858R mutation not detected by solid tumor testing. Conclusions: The magnetic hydrogel particle-based ctDNA extraction technique was highly efficient in recovering ctDNA from plasma. Moreover, the hydrogel particle-based technique required less starting material (1 mL plasma) than column based approaches with similar performance in downstream applications. These results demonstrate the potential benefit of a magnetic hydrogel particle-based ctDNA extraction technique in liquid biopsy testing of NSCLC patients. On-going work is focused on integrating this extraction technique into a multigene liquid biopsy assay.

Nature, Scientific Reports Nanotrap® particles improve detection of SARS-CoV-2 for pooled sample methods, extraction-free saliva methods, and extraction-free transport medium methods Here we present a rapid and versatile method for capturing and concentrating SARS‑CoV‑2 from contrived transport medium and saliva samples using affinity‑capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real‑time RT‑PCR across a range of viral titers (100–1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS‑CoV‑2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS‑CoV‑2 RNA from sample volumes of up to 10 mL.

APPLICATION NOTE SKU 44XXX Download full application note here Key Advantages > Nanotrap® Magnetic Virus Particles concentrate SARS-CoV-2 wastewater sample with no centrifugation or filtration. Introduction According to the Centers for Disease Control and Prevention, SARS-CoV-2 can be shed in the feces of individuals with asymptomatic or symptomatic infections, making SARS-CoV-2 RNA detection in sewage a COVID-19 indicator that is independent of healthcare-seeking behaviors and access to clinical testing.1 Due to low concentrations in water samples such as sewage, SARS-CoV-2 viral particles must be concentrated prior to nucleic acid isolation. The three most common approaches for viral particle concentration are filtration and concentration using electronegative membranes, ultrafiltration, and precipitation. These approaches are time consuming and cumbersome, requiring access to complex laboratory equipment like centrifuges, which limits the daily sample throughput. 2-5 Here we describe a simple and rapid approach, using Nanotrap® Magnetic Virus Particles to capture and concentrate SARS-CoV2 from wastewater samples prior to nucleic acid isolation. Because it requires no filtration or centrifugation steps, this approach is amenable to high throughput implementation.

Poster: Most diagnostic tests for tuberculosis (TB) depend on sputum samples which can be difficult to attain, making the diagnosis of TB in the young children challenging for treating physicians Lipoarabinomannan (LAM) is the only WHO endorsed TB diagnostic biomarker test that can be detected in urine In this study we aim to increase the sensitivity and diagnostic accuracy of one commercially available LAM test by incorporating a processing step using Ceres Nanotrap particles (Ceres Nanosciences, Manassas, VA, USA) to capture and concentrate LAM antigen in urine specimens from children with microbiologically confirmed TB and compare it with age-matched controls. Processing urine with the Ceres Nanotrap particles increased the yield from Alere TB LAM strips from 9% to 54% among children with confirmed TB.

Nature: Scientific Reports volume 10, Article number: 13944 (2020) An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies’ responses was 0.90; 95% CI 0.87–0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77–0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92–0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.

Nature Scientific Reports volume 10, Article number: 19340 (2020) Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

APPLICATION NOTE SKU 44XXX Download full application note here Key Advantages > Nanotrap® Magnetic Virus Particles enable RNA extraction of viral RNA from 4 mL UTM sample volumes in less than 20 minutes, representing a sample extraction method that is roughly 2X faster than column-based methods. > Up to a 4-fold Ct value improvement for large volume pools across a range of viral titers, enabling detection of low viral load samples against a background of 7 negative samples in pooled sample mimics. Introduction As demand increases for testing for SARS-CoV-2, technologies that enable pooled testing for the virus will play a critical role in the efforts to remobilize the economy. Recent analyses from public health experts estimate that pooled testing could lower testing costs by up to three-quarters and could increase testing efficiency by as much as 400%, but concerns remain about reduced test sensitivity associated with pooling. Nanotrap® Virus Particles capture and concentrate multiple strains of influenza, RSV, and coronavirus, thus enhancing detection of those viruses, even in co-infection scenarios. Here, we show that Nanotrap® Magnetic Virus Particles can capture and concentrate heat-inactivated SARS-CoV-2 from large volume universal transport medium samples that simulate patient sample pools. These results suggest that using Nanotrap® particles can alleviate sensitivity concerns associated with pooled sample testing. COVID Coronavirus

APPLICATION NOTE SKU 44XXX Download full application note here Key Advantages > Nanotrap® Magnetic Virus Particles rapidly capture heat-inactivated SARS-CoV-2 from 1 mL of viral transport medium prior to RNA extraction. > Nanotrap® Magnetic Virus Particles improve detection of SARS-CoV-2 using the IDT 2019-nCoV CDC qPCR Probe Assay across 4 logs of viral titers. Introduction Coronaviruses are a large family of viruses that usually cause mild to moderate upper-respiratory tract illnesses in humans. Three times in the 21st century, coronavirus outbreaks have emerged from animal reservoirs to cause severe disease and global transmission concerns, including the most recent outbreak for COVID-19 disease, caused by SARS-CoV-21-3. Nanotrap® Virus Particles have been shown to effectively capture and concentrate multiple strains of influenza, RSV, and coronavirus, thus enhancing detection of those viruses, even in co-infection scenarios4-7. Here, we show Nanotrap® Magnetic Virus Particles can capture and concentrate heat-inactivated SARS-CoV-2 from viral transport medium samples and demonstrate the impact of Nanotrap® particle preconcentration on assay sensitivity across a range of viral titers.

Improving RNA extraction for a more sensitive qRT-PCR assay APPLICATION NOTE SKU 44XXX Download full application note here Key Advantages > Circumvent the low input sample volume restrictions of most extraction methods by capturing influenza A from larger volumes. > Capture and concentrate influenza virus from transport media with Nanotrap particles for greater viral RNA extraction yield and improved qRT-PCR assay sensitivity. Introduction Influenza is responsible for seasonal epidemics each year in the United States that result in an average of 200,000 hospitalizations and tens of thousands of deaths. Accurate and early diagnosis of influenza viral infections are critical for effective treatment and limiting the spread of disease. Nucleic acid-based tests demonstrate high sensitivity and specificity as compared to antigen-based tests; however, limitations on input sample volume can still present sensitivity challenges in complex sample types. Using a simple and efficient pre-concentration workflow in combination with existing molecular test formats can improve detection at earlier time points following infection. Nanotrap Virus Particles can capture, concentrate, and preserve viral pathogens from biological samples and improve downstream diagnostic assays such as enzyme-linked immunosorbent assays (ELISA), next generation sequencing assays (NGS), lateral flow assays (LFA), plaque infectivity assays, and quantitative reverse-transcription polymerase chain reaction assays (qRT-PCR). Here we demonstrate the impact of Nanotrap particle sample processing on influenza virus RNA extraction from viral transport media with QIAGEN’s QIAamp® Viral RNA Mini kit, followed by qRT-PCR detection.