Name: swabbing the urban environment - a pipeline for sampling and detection of sars-cov-2 from environmental reservoirs
Uploaded: 2021-04-09T15:00:00
Duration: 7 min 13 s
Description: Watch this Scientific Journal Video about Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs at JoVE.com

We thank the Viral Information Institute (VII) investigators,&#160;Dr. Anca M. Segall, Willow Segall, Patricia L. Rohwer, Gary Rohwer, Cary L. Rohwer, Magda Silvia Pinetta, Elizabeth Cruz Cano, Dr. Gregory Peters, Dr. Stuart A. Sandin, and Dr. Jennifer Smith for taking the time to collect numerous samples. We also thank Dr. Rob Knight, Dr. Jack Gilbert, Dr. Pedro Balda-Ferre, and Dr. Sarah Allard from the department of Pediatrics at the School of Medicine University of San Diego California (UCSD) for facilitating positive controls and useful feedback. We thank Stacey Carota (SDSU College of Sciences) and Gina Spidel (SDSU) for logistical support and Juan Rodr&#237;guez for the art and graphic design of the sampling protocol. We thank all participants for their commitment and dedication to this project during very difficult times. This work was supported by a generous donation from Dr. Jo Ann Lane (SDSU College of Sciences) and the&#160;National Science Foundation RAPID: Environmental Reservoirs of SARS-CoV-2 grant (Award Number: 2030479).

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
<ol>
	<li>Citizen scientist outreach
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Build a secure web-based sample management platform (SMP) to serve as a database,&#160;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.</li>
		<li>Create a link to the SMP (e.g., https://demo.covidsample.org/)&#160;(Figure 1) for individuals to apply to participate in the environmental sampling effort by answering biosafety-related questions specified in an online form.</li>
		<li>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&#160;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.</li>
		<li>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.</li>
		<li>Include in the sampling section graphic and audiovisual protocols in English and Spanish.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Secure submitted metadata using best practices (e.g., https://demo.covidsample.org/).&#160;</li>
		<li>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 (&#60;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.</li>
		<li>Sanitize all uploaded&#160;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,&#160;and store uploaded images in a separate secure location not accessible to users.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Swab for Corona
	<ol>
		<li>Identify an epidemiologically relevant time window for the sampling effort.</li>
		<li>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).</li>
		<li>Swab rarely disinfected surfaces that are exposed to aerosolized fomites in households and the urban environment.
		<ol>
			<li>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.</li>
			<li>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.</li>
			<li>Swab a surface of 10 cm2. Aided by a toothpick, completely submerge each sample swab in the corresponding pre-labeled tube containing 200 &#181;L of guanidinium thiocyanate solution (GITC). Store tubes at 4 &#176;C until they are transported to the laboratory. Once the samples arrive to&#160;the laboratory, store them at -80 &#176;C. 
			NOTE: GITC is a toxic irritant; avoid contact with skin. GITC solution is simple to prepare from common laboratory chemicals, for the recipe see33,34. It inactivates the virus, stabilizes RNA by denaturing RNases34,35,36, 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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. SARS-CoV-2 detection
<ol>
	<li>Total RNA isolation
	<ol>
		<li>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.</li>
		<li>Thaw samples on ice. Vortex samples for 2 min at medium speed.</li>
		<li>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.</li>
		<li>Pool 50 &#181;L of each of 8 samples into a microcentrifuge tube (total volume 400 &#181;L); save the remaining sample at -80 &#176;C. Add 0.2 volumes (80 &#181;L) of chloroform, vortex for 15 s, and then incubate for 20 min at 4 &#176;C. Centrifuge at 13,000 &#215; g for 20 min at 4 &#176;C.</li>
		<li>Transfer the aqueous (clear liquid) layer into a new microcentrifuge tube. Store the remaining interface and pink liquid in the -80 &#176;C freezer; these fractions contain DNA and proteins33,36.</li>
		<li>Add an equal volume of isopropanol (~200 &#181;L) and 2.6 &#181;L of glycogen coprecipitant (15 mg mL-1)37. Mix well, and incubate at -20 &#176;C for at least 1 h, followed by 4 &#176;C for 10 min to precipitate RNA. 
		&#8203;NOTE: The protocol can be paused here by incubating samples at -20 &#176;C overnight instead of 1 h.</li>
		<li>Centrifuge at 13,000 &#215; g for 20 min at 4 &#176;C. Remove the supernatant without disturbing the pellet. Resuspend the pellet in 50 &#181;L of diethylpyrocarbonate (DEPC)-treated water, and add an equal volume (50 &#181;L) of RNase-free 5 M ammonium acetate and 2.5 volumes (250 &#181;l) of 100% ethanol7,38. 
		NOTE: Ammonium ions inhibit polynucleotide kinase if used in a downstream process38. The mix precipitates RNA while leaving deoxynucleoside triphosphates and oligosaccharides in solution38.</li>
		<li>Mix well, and incubate at -20 &#176;C for at least 1 h, followed by 4 &#176;C for 10 min to precipitate RNA. 
		NOTE: The protocol can be paused here by incubating samples at -20 &#176;C overnight instead of 1 h.</li>
		<li>Centrifuge at 13,000 &#215; g for 20 min at 4 &#176;C. Wash the pellet with 1 mL of cold (-20 &#176;C), freshly made 75% ethanol. Centrifuge at 8,000 &#215; g for 5 min at 4 &#176;C. Remove the supernatant with a P10 pipette to avoid disturbing the pellet.</li>
		<li>Air dry the pellet for 10-15 min until there is no remaining ethanol. Resuspend the pellet in 50 &#181;L of DEPC-treated water, add 5 &#181;L of 10x DNase buffer + 1&#181;L of DNase (2 Units &#181;L-1), and incubate at 37 &#176;C for 30 min.</li>
		<li>Add 0.1 volumes (5.6 &#181;L) of DNase inactivation reagent, incubate at room temperature for 5 min, and mix gently every minute. Centrifuge at 13,000 &#215; g for 2 min, and transfer supernatant to a new tube (~50 &#181;L). Place the tube on ice immediately while preparing the RT-qPCR or RT-LAMP reactions, or store in a -20 &#176;C freezer. 
		NOTE: Perform RNA Isolation in an amplicon-free room to avoid carry-over contamination.</li>
	</ol>
	</li>
	<li>Multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP)
	<ol>
		<li>Prepare a 20X&#160;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 reactions20,28.</li>
		<li>Vortex and spin down the mixture. Dispense 20 &#181;L 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.</li>
		<li>Add 5 &#181;L of RNA to the sample reaction, 5 &#181;L of RNA + 2.5 &#181;L (450 copies) of synthetic SARS-CoV-2 RNA to the spiked reaction, 2.5 &#181;L (450 copies) of synthetic SARS-CoV-2 RNA to the positive control reaction, and 5 &#181;L of H2O to the negative control reaction. Mix well, and spin down the reactions; thaw all RNA on ice.</li>
		<li>While the thermocycler, or the water bath, is heated to 65 &#176;C, allow all reactions to remain at room temperature. The UDG will be&#160;inactivated at &#62;50 &#176;C. Place the reactions in the thermocycler (use heat lid), incubate at 65 &#176;C for 40 min, and let the reactions reach room temperature (~22 &#176;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.&#160;For gel electrophoresis, prepare a 1.5% agarose gel with 1X&#160;DNA gel stain in 0.5% Tris/Borate/EDTA (TBE) buffer. Load 25 &#956;L of the reaction + 5 &#956;L 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).</li>
	</ol>
	</li>
</ol>

All authors declare that no competing interests exist.

Citizen scientist engagement. Citizen scientists were recruited to swab surfaces throughout San Diego County to sample and detect the presence of SARS-CoV-2 in the urban environment. The majority of delivered sampling kits (70%) were returned to the laboratory, and of those, almost all samples were complete (91%) (Figure 3A,B and Table 4). Volunteers could easily request kit delivery/pickup through the web-based platform, and the delivery route-planning software notified citizen scientists of estimated times of arrival, both likely significant factors for the observed success. The average time from when the kit was delivered to the citizen scientist to when it was returned to the laboratory was 8 days, with a median of 3 days and a range of 1-64 days (Figure 3A and Table 4). More frequent reminders to volunteers would likely reduce this lag time.
The data collection platform was successfully used by a great majority of users (73%) (Table 4). While the efforts of the citizen scientists were not measured, field tests showed that the data collection platform significantly reduced the effort and time required to properly complete sample collection. Thus, reducing the amount of bookkeeping&#160;encouraged citizen scientist engagement. The web-based platform intended to overcome demographic limitations by providing a multilingual neural machine translation service and by providing graphic and audiovisual protocols in English and Spanish. This was only partially successful as fewer samples were collected from both the South Bay and North County where most of the county&#39;s Hispanic/Latino population reside45. These areas also harbored 63% (1,700 cases per 100,000) of the total COVID-19 cases in San Diego County with the highest prevalence of the disease46 and rate of hospitalizations (62%)47,48. Although most of the samples came from Central County, a representative number was collected from the most COVID-19-impacted districts and only a small fraction of the samples was positive, which suggests that surface reservoirs of SARS-CoV-2 in the urban environment are relatively rare.
Sample processing. Sampling swabs were wetted with SDS, which inactivated the virus by disrupting its envelope and stabilized the naked RNA by unfolding RNases32. Conveniently during collection, the detergent in the swab cleaned the sampled surface. Environmental samples often contain very small quantities of RNA. To maximize recovery, RNA isolation was performed using a GITC-based, column-free, crude extraction method. GITC, a strong chaotropic agent, disrupts the hydrogen bonds that maintain protein folding (i.e., hydrophobic effect). This action results in the inactivation of viral particles, and the RNA remains stable due to the inhibition of RNAses34,35,36. The GITC solution maintained the stability of the RNA samples without strict cold-chain considerations, which allowed the citizens to maintain the samples at room temperature if a freezer for the provided ice packs was not available. To reduce the potential hazard this reagent poses when direct skin or mucosal contact occurs, citizens were made aware of these risks&#160;by the inclusion of a material safety data sheet provided in the kit&#160;and a warning seal was placed in the box containing the tubes.
The crude GITC-chloroform extraction method aided the recovery of traces of RNA from the swabs, and as shown by the amplification of spiked samples, inhibitors rarely persisted in the samples after extraction. Samples, which were negative for SARS-CoV-2 and showed no RT-LAMP inhibition, represented true negatives or had a lower copy number than the LOD at 100% frequency. Conversely, detection of viral RNA on a surface does not directly imply risk of transmission through contact as the infectivity of the virus from positive samples needs to be tested. Prompt screening of the environment, not limited by the availability of sophisticated supplies or highly qualified personnel, is crucial to assess whether surfaces constitute a viral reservoir and to better direct prevention and containment efforts.
RT-LAMP was selected to be the best method suitable for the proposed detection pipeline. It proved to be a rapid and inexpensive method that was highly resistant to most of the remaining inhibitors and as sensitive and specific as other RT-qPCR methods. Because of their use in clinical settings during the SARS-CoV-2 pandemic, the availability of RT-qPCR kits was impacted by global demand. Moreover, RT-qPCR techniques&#8212;even those formulated to resist inhibitors&#8212;were sensitive to substances contained in the environmental samples collected by a pilot cohort of citizen scientists, even after the use of other common strategies to reduce inhibitor competition for enzyme binding49. These findings are corroborated by a recent study that compared both methods to detect SARS-CoV-2 on swab samples from candy handled by COVID-19 patients and found over 83% result concordance, with 25% lower inhibition in samples analyzed by RT-LAMP15. Furthermore, the GITC-chloroform crude extraction, coupled with RT-LAMP, reduced the cost of reagents and supplies by 42% compared to RNA kit extraction and RT-qPCR (Table of Materials).
This method allowed for high throughput analysis of thousands of surface swab samples. Up to 80 pools, representing 640 samples, were processed in 2&#160;days from RNA extraction to SARS-CoV-2 detection by RT-LAMP. The proposed protocol is semiquantitative,&#160;limited to the detection of viral RNA, and does not indicate the presence of infective viral particles. Further analysis is required to assess the risk of transmission of SARS-CoV-2 from infected fomites present at the swabbed surfaces.
This study presents a protocol to quickly set up a testing strategy that includes an effective workflow when facing a health emergency with a communicable disease.&#160;The proposed sampling protocol is simple and uses supplies commonly found in households, and the viral detection method is carried out on equipment available in basic laboratory settings such as a water bath in lieu of a thermocycler. The costs of RT-LAMP reagents are significantly lower than those needed for RT-qPCR and less susceptible to high global demand scenarios. This study serves as a framework for the assessment of environmental viral reservoirs in future epidemic outbreaks and global pandemics.

SARS-CoV-2 is thought to be mainly transmitted via the inhalation of contaminated aerosols and droplets from direct contact with infected individuals1,2,3,4. However, during the initial phases of the global COVID-19 pandemic, efforts to control transmission of SARS-CoV-2 focused strongly on disinfecting surfaces, handwashing, and sanitization. By the end of 2020, transmission guidelines from the World Health Organization (WHO)5 and the U.S. Centers for Disease Control and Prevention (CDC)6 deemed airborne transmission a hazard mainly when in close contact (&#60;2 m) with an infected person or in the presence of aerosol-generating medical procedures. Self-inoculation after contact with contaminated surfaces or inhalation of aerosolized fomites have yet to be&#160;ruled out as a route of transmission of SARS-CoV-2.
COVID-19 cases have been reported where airborne transmission seems unlikely7,8. SARS-CoV-2 virions remain infectious on copper for up to 8 h, on cardboard and stainless steel for up to 24 h, and on plastic for up to 48 h9. In cruise ship cabins, SARS-CoV-2 RNA was detected 17 days after the passengers had departed7. Air and surface samples from hospitals and mass-transit systems have tested positive for SARS-CoV-2 and other coronaviruses8,10,11,12,13,14. A study performed on the outer packaging of Halloween candy handled by asymptomatic and moderate/mildly symptomatic COVID-19 patients, concluded that the combination of hand washing by the handler and washing of the candy with hand soap reduced SARS-CoV-2 RNA to below threshold levels15.
Several methods for SARS-CoV-2 diagnostics have been published based on real-time reverse-transcription polymerase chain reaction (RT-qPCR)16,17, reverse-transcription loop-mediated isothermal amplification (RT-LAMP)11,18,19,20,21, and CRISPR-Cas technologies18,19,22,23. Most require RNA extraction kits that are often in short supply during periods of significant global demand, and very few have been used for environmental screening of the virus24. The detection of SARS-CoV-2 RNA using RT-LAMP has been demonstrated to be over 83% concordant to using RT-qPCR. Furthermore, RT-LAMP resulted in 25% reduction in inconclusive results compared to RT-qPCR15.
RT-LAMP is a simple technique that uses a reverse transcriptase to synthesize cDNA from an RNA template25, followed by a DNA polymerase with strong strand-displacement activity that synthesizes DNA at constant temperature (i.e., isothermal amplification)26. Higher specificity of viral genome detection is achieved by using four or six primers that recognize six or eight regions of the target DNA. Amplification is initiated from an internal primer and yields a semi-double-stranded DNA structure. The leading strand is then amplified by an outer primer. These amplifications are repeated for the reverse primers. Internal and outer primers on either end have an internal reverse self-complementary&#160;site that forms a loop in the amplification product26,27. In isothermal strand displacement, asynchronous DNA synthesis generates high quantities of amplified product where continuous polymerization amplifies the signal of as few as 10 copies per reaction11,20,28. The colorimetric RT-LAMP mix is weakly buffered and uses phenol red as a pH indicator. As the polymerase incorporates a nucleotide, it releases a proton, and enough protons will change the pH of the solution as well as its color from pink to yellow11,20,28,29.
RT-LAMP was developed for the detection of mosquito-borne diseases in peripheral health care facilities that lack fully equipped laboratories25 and for the rapid detection of other RNA viruses like human immunodeficiency virus30. The most vulnerable populations in epidemic outbreaks&#8212;as per the WHO definition&#8212;often lack sufficient economic resources and the appropriate equipment to carry out detection (United Nations Global Public Health Agenda). In the current SARS-CoV-2 pandemic, supplies such as medical-grade swabs and reagents for RNA extraction kits have not been able to meet global demand, especially in non-manufacturing countries. The proposed protocol used a guanidinium thiocyanate (GITC)-based crude RNA extraction, which effectively preserved the RNA in a cold-chain independent manner and significantly reduced the persistence of inhibitors from the sample. Moreover, the GITC-chloroform extraction protocol is based on the separation of RNA from DNA and proteins followed by the respective precipitation, allowing&#160;recovery of most of the genetic material. These advantages outweigh the potential hazards of citizen scientists handling the chemical if&#160;measures are taken to appropriately inform them of the risks.
The proposed workflow uses materials and reagents that are of general use. It&#160;requires equipment that is available in basic, often rural, laboratory settings. These methods are inexpensive, highly resistant to inhibitors often found in environmental samples or samples that cannot be processed with extraction kits, and eliminate the need for a high-precision thermocycler. This study presents a pipeline for sampling and detection of SARS-CoV-2 from environmental reservoirs on commonly touched and rarely disinfected surfaces of households and the urban environment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SMP, LIMS, and community outreach:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Authentication Application Programming Interface</td>
			<td>Google</td>
			<td>Google Sign-In</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial hosting platform</td>
			<td>GoDaddy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data Charting Application Programming Interface</td>
			<td>Google</td>
			<td>Google Charts</td>
			<td></td>
		</tr>
		<tr>
			<td>Database software</td>
			<td>MySQL&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Delivery route planning software&#160;</td>
			<td>Circuit</td>
			<td>Circuit for Teams</td>
			<td></td>
		</tr>
		<tr>
			<td>Free email service</td>
			<td>Google</td>
			<td>Google Email</td>
			<td></td>
		</tr>
		<tr>
			<td>Geospatial Application Programming Interface</td>
			<td>Google</td>
			<td>Google Maps API</td>
			<td></td>
		</tr>
		<tr>
			<td>Multilingual neural machine translation service</td>
			<td>Google</td>
			<td>Google Translate</td>
			<td></td>
		</tr>
		<tr>
			<td>Online form</td>
			<td>Google</td>
			<td>Google Form</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating system</td>
			<td>Linux</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Web and database development&#160;</td>
			<td>Big Rose Web Design</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Web server software</td>
			<td>Apache&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sampling kit:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coolers</td>
			<td>Coleman (Amazon)</td>
			<td>B00363X3F2</td>
			<td>Cost (US$) per 100 rxns: 70</td>
		</tr>
		<tr>
			<td>Gallon Ziploc bags</td>
			<td>Solimo (Amazon)</td>
			<td>B07BJ495GL</td>
			<td>Cost (US$) per 100 rxns: 18</td>
		</tr>
		<tr>
			<td>Glycerol (hand sanitizer)</td>
			<td>FischerScientific</td>
			<td>G33-4</td>
			<td>Cost (US$) per 100 rxns: 9</td>
		</tr>
		<tr>
			<td>Ice packs</td>
			<td>Ice-Brix (Amazon)</td>
			<td>B075GLD3X1</td>
			<td>Cost (US$) per 100 rxns: 110</td>
		</tr>
		<tr>
			<td>Isopropanol (hand sanitizer)</td>
			<td>FischerScientific</td>
			<td>AA36644K7</td>
			<td>Cost (US$) per 100 rxns: 43</td>
		</tr>
		<tr>
			<td>KN95 masks</td>
			<td>Echo-Sigma</td>
			<td>Echo-Sigma</td>
			<td>Cost (US$) per 100 rxns: 400</td>
		</tr>
		<tr>
			<td>Paper for Protocols and Trizol Safety Sheet</td>
			<td>Office Depot</td>
			<td>348037</td>
			<td>Cost (US$) per 100 rxns: 36</td>
		</tr>
		<tr>
			<td>30 mL spray bottles (SDS and hand sanitizer)</td>
			<td>Anyumocz (Amazon)</td>
			<td>B07T64FHXR</td>
			<td>Cost (US$) per 100 rxns: 80</td>
		</tr>
		<tr>
			<td>RNase, DNase, DNA &#38; PCR inhibitors free Microcentrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>22-281</td>
			<td>Cost (US$) per 100 rxns: 83</td>
		</tr>
		<tr>
			<td>Sample ID solvent resistant labels</td>
			<td>LABTAG</td>
			<td>XST-10C1-1WH</td>
			<td>Cost (US$) per 100 rxns: 68</td>
		</tr>
		<tr>
			<td>Swiffer WetJet pads (swabs)</td>
			<td>Swiffer (Amazon)</td>
			<td>B001F0RBT2</td>
			<td>Cost (US$) per 100 rxns: 8</td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td>Kitchen Essential (Amazon)</td>
			<td>B00PBK4NG6</td>
			<td>Cost (US$) per 100 rxns: 8</td>
		</tr>
		<tr>
			<td>Trizol Reagent (guanidinium isothiocyanate solution - GITC), not LS</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>Cost (US$) per 100 rxns: 40</td>
		</tr>
		<tr>
			<td>Tube boxes</td>
			<td>Genesee Scientific</td>
			<td>21-119</td>
			<td>Cost (US$) per 100 rxns: 180</td>
		</tr>
		<tr>
			<td>Small Ziploc bags</td>
			<td>Ziploc (Amazon)</td>
			<td>B01LRKEI9K</td>
			<td>Cost (US$) per 100 rxns: 8</td>
		</tr>
		<tr>
			<td>Zebra Thermal Transfer Desktop Printer&#160;</td>
			<td>Zebra</td>
			<td>GK420t</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Total Sampling kit Cost (US$) per 100 rxns: 1,160</td>
		</tr>
		<tr>
			<td>Trizol RNA extraction:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate RNase-free</td>
			<td>Invitrogen</td>
			<td>AM9070G</td>
			<td>Cost (US$) per 100 rxns: 2</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>FisherScientific</td>
			<td>C298-500</td>
			<td>Cost (US$) per 100 rxns: 2</td>
		</tr>
		<tr>
			<td>GlycoBlue (glycogen 15 mg/mL)</td>
			<td>Invitrogen</td>
			<td>AM9515</td>
			<td>Cost (US$) per 100 rxns: 80</td>
		</tr>
		<tr>
			<td>Molecular-grade absolute (200 proof) Ethanol</td>
			<td>FisherScientific</td>
			<td>BP2818500</td>
			<td>Cost (US$) per 100 rxns: 30</td>
		</tr>
		<tr>
			<td>Molecular-grade Isopropanol</td>
			<td>FisherScientific</td>
			<td>BP2618500</td>
			<td>Cost (US$) per 100 rxns: 3</td>
		</tr>
		<tr>
			<td>TURBO DNA-free Kit&#160;</td>
			<td>Invitrogen</td>
			<td>AM1907</td>
			<td>Cost (US$) per 100 rxns: 110</td>
		</tr>
		<tr>
			<td>Multiplexed colorimetric RT-LAMP:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine Hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>AAJ6548522</td>
			<td>Cost (US$) per 100 rxns: 1</td>
		</tr>
		<tr>
			<td>RT-LAMP E1-Primers</td>
			<td>IDT</td>
			<td>n/a</td>
			<td>Cost (US$) per 100 rxns: 7</td>
		</tr>
		<tr>
			<td>RT-LAMP N2-Primers</td>
			<td>IDT</td>
			<td>n/a</td>
			<td>Cost (US$) per 100 rxns: 7</td>
		</tr>
		<tr>
			<td>Synthetic SARS-CoV-2 RNA</td>
			<td>ATCC</td>
			<td>VR-3276SD</td>
			<td>Cost (US$) per 100 rxns: 14</td>
		</tr>
		<tr>
			<td>WarmStart Colorimetric LAMP 2X Master Mix with UDG</td>
			<td>NEB</td>
			<td>M1800S</td>
			<td>Cost (US$) per 100 rxns: 210</td>
		</tr>
		<tr>
			<td>Eppendorf Mastercycler Pro Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>950030010</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Total Trizol RNA extraction + LAMP Cost (US$) per 100 rxns: 470</td>
		</tr>
		<tr>
			<td>Kit for RNA extraction:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DSP Viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>61904</td>
			<td>Cost (US$) per 100 rxns: 570</td>
		</tr>
		<tr>
			<td>RT-qPCR:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic SARS-CoV-2 RNA</td>
			<td>ATCC</td>
			<td>VR-3276SD</td>
			<td>Cost (US$) per 100 rxns: 14</td>
		</tr>
		<tr>
			<td>TaqMan Fast Virus 1-Step Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4444432</td>
			<td>Cost (US$) per 100 rxns: 180</td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) N1,N2 Primers and Probes</td>
			<td>IDT</td>
			<td>10006713</td>
			<td>Cost (US$) per 100 rxns: 20</td>
		</tr>
		<tr>
			<td>qScript XLT 1-Step RT-qPCR ToughMix</td>
			<td>Quantabio</td>
			<td>95132-100</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiNova Pathogen&#160;</td>
			<td>Qiagen</td>
			<td>208652</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiNova Probe</td>
			<td>Qiagen</td>
			<td>208352</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPlex 1-Step ToughMix&#160;</td>
			<td>Quantabio</td>
			<td>95166-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>BioRad</td>
			<td>1855196</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Kit for RNA extraction + RT-qPCR Cost (US$) per 100 rxns: 790&#160;</td>
		</tr>
		<tr>
			<td>RT-PCR:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV One-Step RT-PCR&#160;</td>
			<td>Invitrogen</td>
			<td>12594025</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab cleanup:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNAZap</td>
			<td>Invitrogen</td>
			<td>AM9890</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAZap</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td></td>
		</tr>
	</tbody>
</table>

swabbing the urban environment - a pipeline for sampling and detection of sars-cov-2 from environmental reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

Watch this Scientific Journal Video about Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs at JoVE.com

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

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.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Research

JoVE Journal

Biology

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...