Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs
April 9th, 2021
A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.
This study presents a novel workflow to detect SARS-CoV-2 on surfaces that are rarely cleaned in the urban environment, such as gas pump handles, playgrounds, and ATMs. In a pandemic, supplies are scarce. We use easily obtainable materials and reagents and equipment available in basic laboratory settings.
We use an extraction method that preserves the RNA without a cold chain, and a detection method resistant to inhibitors. This protocol is of public health interest. It provides a framework for the assessment of environmental viral reservoirs for the current COVID-19 pandemic and other infectious agents during future global outbreaks.
Recruit citizen scientists using a direct and clear call to action released via local and social media. Create a social media handle to connect the topic across social media content. Create a link to the SMP, providing a multi-lingual plug-in to enable navigation in multiple languages for individuals to apply to participate in the environmental sampling effort by answering biosafety related questions specified in an online form.
Include in the sampling section graphic and audiovisual protocols in English and Spanish. Visualize geospatial data using a geospatial application programming interface facilitated by a cloud computer service provider. Store the data submitted to the LIMS through the SMP to facilitate centralized storage, tracking of processing workflows, and management of the logistics.
Pre-load information such as sample kit ID, sample ID, date, time, and global positioning system coordinates to enable data type compliance and minimize the error. Include a sample pickup request link for the participants, which they can use once they have collected all samples. Build a kit that contains all the sampling supplies, including the necessary personal protective equipment, like mask and gloves, a sampling protocol, and biosafety relevant information.
Swab rarely disinfected surfaces in households and the urban environment by wetting a one centimeter square polyester absorbent swab with a detergent and swabbing a surface of 10 centimeters squared. Aided by a toothpick, submerge each sample swab in the pre-labeled tube containing 200 microliters of guanidinium thiocyanate. Wear the provided mask and a new pair of gloves for the collection of each sample to avoid cross-contamination.
After finishing the sampling, use the provided hand sanitizer. Store the tubes at four degrees Celsius until they are transported to the laboratory. Once the samples arrive in the laboratory, store them at minus 80 degrees Celsius.
To increase the speed of the screening, process the samples in pools. If a pool is positive, extract the RNA of each sample independently. Combine the samples from each sampling kit into two pools by pooling 50 microliters of each of the eight samples into a microcentrifuge tube and saving the remaining samples at minus 80 degrees Celsius.
Add 80 microliters of chloroform and vortex for 15 seconds. Then incubate for 20 minutes at four degrees Celsius. Centrifuge at 13, 000 times G for 20 minutes at four degrees Celsius.
Transfer the aqueous layer into a new microcentrifuge tube. Store the remaining interface and pink liquid in the minus 80 degree Celsius freezer. These fractions contain DNA and proteins.
Extract RNA from the recovered aqueous layer using a guanidinium thiocyanate based RNA crude extraction protocol. Prepare the RT-LAMP reaction mix at room temperature with 10%excess volume to account for pipetting loss. Add five microliters of RNA to the sample reaction and five microliters of RNA plus 2.5 microliters of synthetic SARS-CoV-2 RNA to the spiked reaction.
Add 2.5 microliters of synthetic SARS-CoV-2 RNA to the positive control and five microliters of water to the negative control. Mix well and spin down the reactions. For colorimetric observation, a negative result is indicated by pink and a positive result is indicated by yellow.
After RT-LAMP, perform gel electrophoresis. The distribution of sample collection sites is shown here. A majority of kits were complete, and the corresponding data was uploaded to the LIMS.
The limit of detection at a frequency of 100%was 500 copies per 25 microliters of reaction. In the colorimetric RT-LAMP, positive samples changed color from pink to yellow due to a pH shift from eight to 5.5. At low copy numbers, samples were run on an agarose gel to confirm the positives with the resulting ladder-like pattern.
RT QPCR methods were tested with environmental samples. All master mixes were sensitive to inhibitors at low copy number concentrations of the positive control. At low concentrations of the template, traditional RT PCR methods showed false positives and false negatives.
Lastly, a technique called rolling circle amplification detected small quantities of the target sequence. However, it showed amplification of the probe in the absence of an RNA template. It is vital to release a call to action that reaches all sectors of the community so the sampling truly represents the risk of exposure of all members of that community.
This framework detects SARS-CoV-2 genetic material. Studies to test viral viability are the next step.
Overview
This article presents a citizen science-based workflow for detecting SARS-CoV-2 on rarely cleaned urban surfaces during the COVID-19 pandemic. The protocol leverages easily accessible materials and reagents, enabling large-scale environmental sampling and sensitive detection of viral RNA, even under supply chain constraints. The approach is designed to be robust against inhibitors commonly found in environmental samples and is adaptable for future public health surveillance of other viruses.
Key Study Components
Area of Science
- Environmental virology
- Public health surveillance
- Molecular diagnostics
Background
- Surface transmission of SARS-CoV-2 was a concern during the COVID-19 pandemic.
- Most environmental surveillance focused on high-prevalence locations like hospitals and public transport.
- Urban surfaces that are rarely cleaned may serve as viral reservoirs.
- Supply shortages during the pandemic necessitated alternative sampling and detection strategies.
Purpose of Study
- To develop and validate a workflow for detecting SARS-CoV-2 on rarely disinfected urban surfaces.
- To engage citizen scientists in large-scale environmental sampling.
- To create a protocol resilient to supply shortages and environmental inhibitors.
Methods Used
- Recruitment of 350 citizen scientists via local and social media, with multilingual support.
- Distribution of sampling kits containing PPE, swabs, and protocols.
- Swabbing of 10 cm2 surface areas using detergent-wetted polyester swabs.
- Sample preservation in guanidinium thiocyanate without cold chain requirements.
- Pooling of samples for initial screening, followed by individual RNA extraction if pools tested positive.
- RNA extraction using a guanidinium thiocyanate-based protocol.
- Detection via colorimetric RT-LAMP, confirmed by gel electrophoresis.
- Comparison with RT-qPCR and rolling circle amplification methods.
- Centralized data management using a Laboratory Information Management System (LIMS) and geospatial visualization tools.
Main Results
- Over 4,000 samples were collected from diverse urban surfaces.
- The RT-LAMP assay demonstrated a detection limit of 500 copies per 25 µL reaction with clear colorimetric readout.
- Pooling increased screening efficiency; positive pools were deconvoluted for individual analysis.
- RT-qPCR was sensitive to inhibitors at low copy numbers, leading to false positives/negatives.
- Rolling circle amplification showed non-specific amplification in the absence of RNA template.
- The workflow successfully engaged the community and enabled comprehensive environmental surveillance.
Conclusions
- The described protocol enables sensitive detection of SARS-CoV-2 RNA on urban surfaces using accessible materials and citizen participation.
- The workflow is robust against environmental inhibitors and adaptable for future outbreaks of other viruses.
- Further studies are needed to assess viral viability on detected surfaces.
Frequently Asked Questions
Executive Industry Relevance
Environmental surveillance for viral RNA on urban surfaces provides a scalable framework for early detection of community transmission risks and environmental reservoirs. This workflow enables rapid, distributed sampling and robust detection even under supply chain constraints, supporting predictive confidence in public health and biopharma R&D. The approach is adaptable for future pathogen monitoring, informing risk assessment and intervention strategies across the discovery-to-surveillance continuum.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables hypothesis-driven assessment of environmental viral reservoirs relevant to transmission dynamics.
- Supports biological de-risking by clarifying the presence and distribution of viral RNA on diverse surfaces.
- Facilitates predictive confidence in environmental exposure models for infectious agents.
Screening & Assay Development
- Provides validated protocols for high-sensitivity detection of viral RNA in complex environmental matrices.
- Standardizes sample collection and processing for reproducible, quantitative outputs across large cohorts.
- Demonstrates assay robustness against inhibitors, supporting reliable screening in real-world conditions.
Translational & Preclinical Research
- Aligns environmental surveillance data with public health biomarker strategies for outbreak preparedness.
- Enables continuity from environmental detection to preclinical risk modeling and intervention planning.
- Supports risk-adjusted advancement of surveillance tools for emerging pathogens.
Pipeline & Workflow Integration
This workflow bridges environmental sampling, molecular detection, and centralized data management, positioning it from early discovery through translational surveillance.
- Discovery Biology: Supports hypothesis testing on environmental transmission and reservoir mapping.
- Screening: Delivers reproducible, quantitative detection of viral RNA from pooled and individual samples.
- Analytics: Integrates geospatial and LIMS-based data for comparative analysis and compliance.
- Translational Research: Informs public health biomarker alignment and outbreak response strategies.
- Enterprise Reuse: Establishes a scalable, adaptable platform for future pathogen surveillance initiatives.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in environmental risk assessment and target validation.
- Operational Value: Enables standardized, scalable sampling and detection under resource constraints.
- Strategic Value: Improves go/no-go decisions for surveillance deployment and intervention prioritization.
- Portfolio Impact: Supports risk-adjusted prioritization of surveillance and diagnostic tool development.
Implementation Considerations
- Requires expertise in molecular diagnostics and environmental sampling protocols.
- Needs access to basic laboratory infrastructure and LIMS for data management.
- Demands cross-team standardization for sample collection, processing, and data reporting.
- Adaptable to various urban and community environments with minimal supply requirements.
- Detection limited to viral RNA; viability studies are a necessary next step.
