This study presents a novel workflow to detect SARS-CoV-2 on surfaces that are rarely cleaned in the urban environment, such as gas pump handles, playgrounds, and ATMs. In a pandemic, supplies are scarce. We use easily obtainable materials and reagents and equipment available in basic laboratory settings.
We use an extraction method that preserves the RNA without a cold chain, and a detection method resistant to inhibitors. This protocol is of public health interest. It provides a framework for the assessment of environmental viral reservoirs for the current COVID-19 pandemic and other infectious agents during future global outbreaks.
Recruit citizen scientists using a direct and clear call to action released via local and social media. Create a social media handle to connect the topic across social media content. Create a link to the SMP, providing a multi-lingual plug-in to enable navigation in multiple languages for individuals to apply to participate in the environmental sampling effort by answering biosafety related questions specified in an online form.
Include in the sampling section graphic and audiovisual protocols in English and Spanish. Visualize geospatial data using a geospatial application programming interface facilitated by a cloud computer service provider. Store the data submitted to the LIMS through the SMP to facilitate centralized storage, tracking of processing workflows, and management of the logistics.
Pre-load information such as sample kit ID, sample ID, date, time, and global positioning system coordinates to enable data type compliance and minimize the error. Include a sample pickup request link for the participants, which they can use once they have collected all samples. Build a kit that contains all the sampling supplies, including the necessary personal protective equipment, like mask and gloves, a sampling protocol, and biosafety relevant information.
Swab rarely disinfected surfaces in households and the urban environment by wetting a one centimeter square polyester absorbent swab with a detergent and swabbing a surface of 10 centimeters squared. Aided by a toothpick, submerge each sample swab in the pre-labeled tube containing 200 microliters of guanidinium thiocyanate. Wear the provided mask and a new pair of gloves for the collection of each sample to avoid cross-contamination.
After finishing the sampling, use the provided hand sanitizer. Store the tubes at four degrees Celsius until they are transported to the laboratory. Once the samples arrive in the laboratory, store them at minus 80 degrees Celsius.
To increase the speed of the screening, process the samples in pools. If a pool is positive, extract the RNA of each sample independently. Combine the samples from each sampling kit into two pools by pooling 50 microliters of each of the eight samples into a microcentrifuge tube and saving the remaining samples at minus 80 degrees Celsius.
Add 80 microliters of chloroform and vortex for 15 seconds. Then incubate for 20 minutes at four degrees Celsius. Centrifuge at 13, 000 times G for 20 minutes at four degrees Celsius.
Transfer the aqueous layer into a new microcentrifuge tube. Store the remaining interface and pink liquid in the minus 80 degree Celsius freezer. These fractions contain DNA and proteins.
Extract RNA from the recovered aqueous layer using a guanidinium thiocyanate based RNA crude extraction protocol. Prepare the RT-LAMP reaction mix at room temperature with 10%excess volume to account for pipetting loss. Add five microliters of RNA to the sample reaction and five microliters of RNA plus 2.5 microliters of synthetic SARS-CoV-2 RNA to the spiked reaction.
Add 2.5 microliters of synthetic SARS-CoV-2 RNA to the positive control and five microliters of water to the negative control. Mix well and spin down the reactions. For colorimetric observation, a negative result is indicated by pink and a positive result is indicated by yellow.
After RT-LAMP, perform gel electrophoresis. The distribution of sample collection sites is shown here. A majority of kits were complete, and the corresponding data was uploaded to the LIMS.
The limit of detection at a frequency of 100%was 500 copies per 25 microliters of reaction. In the colorimetric RT-LAMP, positive samples changed color from pink to yellow due to a pH shift from eight to 5.5. At low copy numbers, samples were run on an agarose gel to confirm the positives with the resulting ladder-like pattern.
RT QPCR methods were tested with environmental samples. All master mixes were sensitive to inhibitors at low copy number concentrations of the positive control. At low concentrations of the template, traditional RT PCR methods showed false positives and false negatives.
Lastly, a technique called rolling circle amplification detected small quantities of the target sequence. However, it showed amplification of the probe in the absence of an RNA template. It is vital to release a call to action that reaches all sectors of the community so the sampling truly represents the risk of exposure of all members of that community.
This framework detects SARS-CoV-2 genetic material. Studies to test viral viability are the next step.
