Swabbing the Urban Environment – A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

See the Table of Materials for a detailed list of reagents and supplies, including catalog numbers, manufacturer, and corresponding costs.

1. Sampling the urban environment

1. Citizen scientist outreach
   1. Recruit citizen scientists using a direct and clear call-to-action released via local and social media. Create a social media handle (e.g., #swab4corona) to connect the topic across social media content.
   2. Create an email account for direct communication between the laboratory team and each citizen scientist, managed by a person fluent in the main languages of the region of interest (e.g., Spanish and English for San Diego County).
   3. Build a secure web-based sample management platform (SMP) to serve as a database, a laboratory information management system (LIMS), and to communicate with citizen scientists.
      NOTE: The SMP provides a centralized location where users request a kit, access sample collection protocols, submit sample metadata, and request a pickup for completed sample kits.
   4. Create a link to the SMP (e.g., https://demo.covidsample.org/) (Figure 1) for individuals to apply to participate in the environmental sampling effort by answering biosafety-related questions specified in an online form.
   5. Secure access to the SMP using an Authentication Application Programming Interface facilitated by a cloud computer service provider. Give approved users access to the SMP.
      NOTE: Refer to literature for a description of the OAuth 2.0 protocol³¹ for authentication and authorization. It provides a frictionless sign-in process for citizen scientist volunteers. It also allows users to sign-in with an existing account, eliminating the need to create a custom sign-in solution and manage user credentials, which saves a significant amount of time and encourages participation. According to recent reports, the free email service available for the chosen cloud computer service provider has approximately 1.5 billion monthly-active email users; requiring an email account from this service provider for participation is not considered a discouraging factor.
   6. Explain the objective of the study and biosafety considerations to the citizen scientists at the SMP before they request their first kit. Provide a multilingual plugin to enable navigation in any of the languages available from a multilingual neural machine translation service facilitated by a cloud computer service provider.
   7. Include in the sampling section graphic and audiovisual protocols in English and Spanish.
   8. Assign a unique identifier to each kit, and design the user interface to utilize buttons linked to Sample ID to streamline the data entry process (Figure 1A).
   9. Use a delivery route planning software with a mobile-device application to be used by drivers to optimize delivery/pickup routes and notify citizen scientists of accurate estimated times of arrival.
   10. Build the LIMS platform on a PHP web service stack, and host it on a commercial hosting platform (suggested operating system, web server software, and database software are specified in the Table of Materials).
   11. Provide a secure web-based application interface to enable lab personnel to manage data quickly and easily in the LIMS. Provide data visualization using a data Charting Application Programming Interface facilitated by a cloud computer service provider.
   12. 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 (1) centralized storage of project data; (2) tracking of sample/data processing workflows; and (3) management of the logistics of sample kit distribution to citizen scientists.
   13. Secure submitted metadata using best practices (e.g., https://demo.covidsample.org/). 
   14. Pre-load information such as Sample Kit ID, Sample ID, date, time, and Global Positioning System (GPS) coordinates (automatically gathered from a picture of the site) to enable data-type compliance and minimize the submission of erroneous or missing data by the user (Figure 1B). Include the following fields to be manually and swiftly (<1 min) filled by the citizen scientist: date and time of collection, a brief description of the location, and a picture of the sampling site.
   15. Sanitize all uploaded data, and validate for data type. For example, validate image data uploaded by users to select .jpg files, rename them with Sample ID for fast association with the sample, and store uploaded images in a separate secure location not accessible to users.
   16. Activate the option to request kit delivery and pickup when all samples (16) have been completed. Additionally, activate the option to request a new kit to be delivered upon pickup of the previous one (Figure 1A).
       NOTE: For volunteers who prefer a non-web-based platform and for those worried about disclosing their GPS location (e.g., members of the community concerned about their migratory status), kits can be delivered at an agreed meeting location and volunteers asked to record a written version of the data collection. For communication between the laboratory and each citizen scientist, have a bilingual member of the project available for telephone calls and texts.
2. Swab for Corona
   1. Identify an epidemiologically relevant time window for the sampling effort.
   2. Build a kit that contains all the sampling supplies, including the necessary personal protective equipment (i.e., mask, gloves), a sampling protocol, and biosafety relevant information (Figure 2). Pre-label each tube with the assigned unique identifier (Sample ID).
   3. Swab rarely disinfected surfaces that are exposed to aerosolized fomites in households and the urban environment.
      1. Wear the provided mask in public 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.
      2. Wet a 1 cm² polyester absorbent swab (e.g., mop pads) with a detergent (e.g., 0.5% sodium dodecyl sulfate (SDS)) to inactivate the virus by disrupting its envelope and to stabilize the naked RNA by inducing unfolding of RNases³².
      3. Swab a surface of 10 cm². Aided by a toothpick, completely submerge each sample swab in the corresponding pre-labeled tube containing 200 µL of guanidinium thiocyanate solution (GITC). Store tubes at 4 °C until they are transported to the laboratory. Once the samples arrive to the laboratory, store them at -80 °C.
         NOTE: GITC is a toxic irritant; avoid contact with skin. GITC solution is simple to prepare from common laboratory chemicals, for the recipe see^33,34. It inactivates the virus, stabilizes RNA by denaturing RNases^34,35,36, and stabilizes samples at room temperature. The kit, however, includes ice packs to keep the samples cold without the need to use household refrigerators for storage.

2. SARS-CoV-2 detection

1. Total RNA isolation
   1. Disinfect surfaces, equipment, and pipettors with a solution of 2 mM copper sulfate and 3% hydrogen peroxide; followed by a solution of 10% bleach, 90 mM sodium bicarbonate, 5% SDS, and 2.5% NaOH. Wipe thoroughly with distilled water followed by 75% ethanol.
      NOTE: These solutions are an alternative to the commercially available solutions.
   2. Thaw samples on ice. Vortex samples for 2 min at medium speed.
   3. To increase the speed of the screening, process the samples in pools. If a pool is positive, extract the RNA of each sample independently to find the positive sample/s. Combine the samples from each sampling kit (16 total) into 2 pools of 8 samples.
      NOTE: Having 8 samples per pool means that only 2 pools need to be processed per kit. If a pool is positive, then the individual samples are re-processed for individual RT-LAMP analysis. This reduces time, costs, and reagents.
   4. Pool 50 µL of each of 8 samples into a microcentrifuge tube (total volume 400 µL); save the remaining sample at -80 °C. Add 0.2 volumes (80 µL) of chloroform, vortex for 15 s, and then incubate for 20 min at 4 °C. Centrifuge at 13,000 × g for 20 min at 4 °C.
   5. Transfer the aqueous (clear liquid) layer into a new microcentrifuge tube. Store the remaining interface and pink liquid in the -80 °C freezer; these fractions contain DNA and proteins^33,36.
   6. Add an equal volume of isopropanol (~200 µL) and 2.6 µL of glycogen coprecipitant (15 mg mL^-1)³⁷. Mix well, and incubate at -20 °C for at least 1 h, followed by 4 °C for 10 min to precipitate RNA.
      ​NOTE: The protocol can be paused here by incubating samples at -20 °C overnight instead of 1 h.
   7. Centrifuge at 13,000 × g for 20 min at 4 °C. Remove the supernatant without disturbing the pellet. Resuspend the pellet in 50 µL of diethylpyrocarbonate (DEPC)-treated water, and add an equal volume (50 µL) of RNase-free 5 M ammonium acetate and 2.5 volumes (250 µl) of 100% ethanol^7,38.
      NOTE: Ammonium ions inhibit polynucleotide kinase if used in a downstream process³⁸. The mix precipitates RNA while leaving deoxynucleoside triphosphates and oligosaccharides in solution³⁸.
   8. Mix well, and incubate at -20 °C for at least 1 h, followed by 4 °C for 10 min to precipitate RNA.
      NOTE: The protocol can be paused here by incubating samples at -20 °C overnight instead of 1 h.
   9. Centrifuge at 13,000 × g for 20 min at 4 °C. Wash the pellet with 1 mL of cold (-20 °C), freshly made 75% ethanol. Centrifuge at 8,000 × g for 5 min at 4 °C. Remove the supernatant with a P10 pipette to avoid disturbing the pellet.
   10. Air dry the pellet for 10-15 min until there is no remaining ethanol. Resuspend the pellet in 50 µL of DEPC-treated water, add 5 µL of 10x DNase buffer + 1µL of DNase (2 Units µL^-1), and incubate at 37 °C for 30 min.
   11. Add 0.1 volumes (5.6 µL) of DNase inactivation reagent, incubate at room temperature for 5 min, and mix gently every minute. Centrifuge at 13,000 × g for 2 min, and transfer supernatant to a new tube (~50 µL). Place the tube on ice immediately while preparing the RT-qPCR or RT-LAMP reactions, or store in a -20 °C freezer.
       NOTE: Perform RNA Isolation in an amplicon-free room to avoid carry-over contamination.
2. Multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP)
   1. Prepare a 20X primer mix solution (Table 1) for each set of primers (Table 2). Prepare the RT-LAMP reaction mix (Table 3) at room temperature with 10% excess volume to account for pipetting loss.
      NOTE: The colorimetric LAMP 2X master mix with Antarctic thermolabile uracil-DNA glycosylase (UDG) prevents amplification of DNA contamination from previous reactions^20,28.
   2. Vortex and spin down the mixture. Dispense 20 µL of the mix into each reaction tube: sample, spiked, positive control, and negative control. Incubate the reactions in the tubes at room temperature for 10 min to allow the UDG to act on potential carry-over contamination.
   3. Add 5 µL of RNA to the sample reaction, 5 µL of RNA + 2.5 µL (450 copies) of synthetic SARS-CoV-2 RNA to the spiked reaction, 2.5 µL (450 copies) of synthetic SARS-CoV-2 RNA to the positive control reaction, and 5 µL of H₂O to the negative control reaction. Mix well, and spin down the reactions; thaw all RNA on ice.
   4. While the thermocycler, or the water bath, is heated to 65 °C, allow all reactions to remain at room temperature. The UDG will be inactivated at >50 °C. Place the reactions in the thermocycler (use heat lid), incubate at 65 °C for 40 min, and let the reactions reach room temperature (~22 °C for 5 min), or cool on ice for 1 min. Analyze the results using the colorimetric feature (simple observation) or by running the products in an agarose gel.
      NOTE: Although the limit-of-detection (LOD) is 10 copies per reaction, the frequency of detection increases as the copy number approaches 500 copies per reaction (Figure 3A). For colorimetric observation, note that a negative result is indicated by pink (pH = 8.8), whereas a positive result is indicated by yellow (pH = 5) (Figure 3B). The colorimetric option avoids the opening of the RT-LAMP tubes after amplification, which will reduce the volume of the RT-LAMP products in the working environment and carry-over contamination. For gel electrophoresis, prepare a 1.5% agarose gel with 1X DNA gel stain in 0.5% Tris/Borate/EDTA (TBE) buffer. Load 25 μL of the reaction + 5 μL of 6X loading dye in each well. Run the gel at 100 V for 60 min. A molecular marker is not needed as positive samples show a ladder pattern (Figure 3C).