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><tbody><tr><td>&lt;strong&gt;SMP, LIMS, and community outreach:&lt;/strong&gt;</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&amp;nbsp;</td><td></td><td></td></tr><tr><td>Delivery route planning software&amp;nbsp;</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&amp;nbsp;</td><td>Big Rose Web Design</td><td></td><td></td></tr><tr><td>Web server software</td><td>Apache&amp;nbsp;</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Sampling kit:&lt;/strong&gt;</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 &amp;amp; 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$) per 100 rxns: 8</td></tr><tr><td>Zebra Thermal Transfer Desktop Printer&amp;nbsp;</td><td>Zebra</td><td>GK420t</td><td></td></tr><tr><td colspan="4">Total Sampling kit Cost (US$) per 100 rxns: 1,160</td></tr><tr><td>&lt;strong&gt;Trizol RNA extraction:&lt;/strong&gt;</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$) per 100 rxns: 3</td></tr><tr><td>TURBO DNA-free Kit&amp;nbsp;</td><td>Invitrogen</td><td>AM1907</td><td>Cost (US$) per 100 rxns: 110</td></tr><tr><td>&lt;strong&gt;Multiplexed colorimetric RT-LAMP:&lt;/strong&gt;</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$) per 100 rxns: 210</td></tr><tr><td>Eppendorf Mastercycler Pro Thermal Cycler</td><td>Eppendorf</td><td>950030010</td><td></td></tr><tr><td colspan="4">Total Trizol RNA extraction + LAMP Cost (US$) per 100 rxns: 470</td></tr><tr><td>&lt;strong&gt;Kit for RNA extraction:&lt;/strong&gt;</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$) per 100 rxns: 570</td></tr><tr><td>&lt;strong&gt;RT-qPCR:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Synthetic SARS-CoV-2 RNA</td><td>ATCC</td><td>VR-3276SD</td><td>Cost (US$) 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&amp;nbsp;</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&amp;nbsp;</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 colspan="4">Kit for RNA extraction + RT-qPCR Cost (US$) per 100 rxns: 790&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;RT-PCR:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SuperScript IV One-Step RT-PCR&amp;nbsp;</td><td>Invitrogen</td><td>12594025</td><td></td></tr><tr><td>&lt;strong&gt;Lab cleanup:&lt;/strong&gt;</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 ...

swabbing-urban-environment-pipeline-for-sampling-detection-sars-cov-2

Nanyang Technological University

Research

JoVE Journal

Biology

4.2K Views.  San Diego State University. Deze studie presenteert een nieuwe workflow om SARS-CoV-2 te detecteren op oppervlakken die zelden worden gereinigd in de stedelijke omgeving, zoals gaspompgrepen, speeltuinen en geldautomaten. In een pandemie zijn voorraden schaars. We gebruiken gemakkelijk verkrijgbare materialen en reagentia en apparatuur die beschikbaar zijn in basislaboratoriuminstellingen.We gebruiken een extractiemethode die het RNA behoudt zonder koudeketen en een detectiemethode die bestand is tegen remmers. D...

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

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

The protocol is intended to serve as a blueprint for universities and other organizations considering large-scale testing for SARS-CoV-2 or developing preparedness plans for future viral outbreaks.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

Genetics

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Environment

Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

Wastewater-based epidemiology has emerged as a promising and efficacious surveillance system for SARS-CoV-2 and other infectious diseases in many nations. The process typically involves wastewater concentration, nucleic acid extraction, amplification of selected genomic segments, and detection and quantification of the amplified genomic segment. This methodology can similarly be leveraged to detect and quantify infectious agents, such as SARS-CoV-2, in air samples. Initially, SARS-CoV-2 was presumed to spread primarily through close personal contact with droplets generated by an infected individual while speaking, sneezing, coughing, singing, or breathing. However, a growing number of studies have reported the presence of SARS-CoV-2 RNA in the air of healthcare facilities, establishing airborne transmission as a viable route for the virus. This study presents a composite of established protocols to facilitate environmental detection, quantification, and sequencing of viruses from both wastewater and air samples.

This protocol aims to quantify SARS-CoV-2 RNA in wastewater and air samples to be used for wastewater-based epidemiology studies and to assess the exposure risk to SARS-CoV-2 in indoor and outdoor aerosols. This protocol also describes a tiled amplicon long-template sequencing approach for SARS-CoV-2 whole genome characterization.

Wastewater-based epidemiology has emerged as a promising and efficacious surveillance system for SARS-CoV-2 and other infectious diseases in many nations. ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Immunology and Infection

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 nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

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 Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

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

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

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) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

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

Using Colorimetric and RT-LAMP Analysis to Detect SARS-CoV-2 

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern ...

Multiplex RT-qPCR Assay for Respiratory Virus Detection

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

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

Samenvatting

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