key: cord-0740689-775b3i10
authors: Hoffman, Jason S.; Hirano, Matthew; Panpradist, Nuttada; Breda, Joseph; Ruth, Parker; Xu, Yuanyi; Lester, Jonathan; Nguyen, Bichlien H.; Ceze, Luis; Patel, Shwetak N.
title: Passively sensing SARS-CoV-2 RNA in public transit buses
date: 2022-01-08
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.152790
sha: 48537bcbffba3943f4680e8fcdbb31b0ae1aeb82
doc_id: 740689
cord_uid: 775b3i10

Affordably tracking the transmission of respiratory infectious diseases in urban transport infrastructures can inform individuals about potential exposure to diseases and guide public policymakers to prepare timely responses based on geographical transmission in different areas in the city. Towards that end, we designed and tested a method to detect SARS-CoV-2 RNA in the air filters of public buses, revealing that air filters could be used as passive fabric sensors for the detection of viral presence. We placed and retrieved filters in the existing HVAC systems of public buses to test for the presence of trapped SARS-CoV-2 RNA using phenol-chloroform extraction and RT-qPCR. SARS-CoV-2 RNA was detected in 14% (5/37) of public bus filters tested in Seattle, Washington, from August 2020 to March 2021. These results indicate that this sensing system is feasible and that, if scaled, this method could provide a unique lens into the geographically relevant transmission of SARS-CoV-2 through public transit rider vectors, pooling samples of riders over time in a passive manner without installing any additional systems on transit vehicles.

Therefore, they represent an opportunity for passive sensing if viral presence can be detected in the system. Prior work has examined risk of transmission for passengers on buses, trains, and airplanes at local, national, and international scale [27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39] ;

however, prior work has not explored community monitoring on public transit. One potential reason for this is the cost and time associated with known sampling methods with adequate Limits of Detection (LOD) to sense the low number of copies of virus expected in filters without employing active systems of collection, such as environmental swabbing or vacuum-like Personal

Environmental Monitor (PEM) equipment [40] . Rapid and inexpensive RNA extraction methods have detected 10-20 copies/reaction, which may be above the viral copies recovered from passive HVAC systems in non-concentrated settings outside of hospitals [41] . Additionally, virus particles can remain viable for 7 days on porous surfaces, like air filters, and 3 days on non-porous surfaces, like metal hand-grips [42, 43] . Therefore, air filters may accumulate and maintain virus over a longer time than swabbed surfaces, capturing data from more individuals with a single sample, 

Between August 2020 and March 2021, environmental samples were collected from 15 actively deployed buses in the Seattle King County Metro fleet ( Figure 1A ). Bus selection was narrowed down to the main bus depot that serviced the Downtown Seattle area, which has the highest ridership. Individual buses were selected to be sampled via a convenience sampling approach based on which buses could be made available at the depot on a regular basis between 7:00-9:00 AM for sample retrieval. All bagged samples were placed in a plastic secondary container, which was wiped with bleach-based disinfectant, and transported to an approved lab facility. All procedures involving the untreated filter material were performed in a BSL2-certified Class II A2 biosafety cabinet. All types of filters that were used are shown in Figure 1C . Due to safety-related lab space and chemical SOP limits for phenol-chloroform isoamyl extraction, a maximum of n=6 buses (2 replicates for J o u r n a l P r e -p r o o f 

Sample extraction for testing was performed within the same day of the sample collection from metro buses. Detection of SARS-CoV-2 RNA consisted of the following steps: viral extraction and lysis, RNA isolation via phenol-chloroform isoamyl extraction, and RNA detection via RT-qPCR ( Figure 1A ).

Filters collected from buses were cut into 2-cm 2 pieces. Two pieces, considered sample replicates, were placed into microcentrifuge tubes containing 200 µL lysis buffer (50mM EDTA pH 8.0, 250mM Tris-HCl pH 8.0, 50mM NaCl, 1% (w/v) SDS) [47] . The tubes were placed on a foam tube rack attached to a vortexer and agitated for 15 minutes, at high speed, at room temperature. After vortexing, 600 µL TRIzol was added to each tube, pipette-mixed 10 times, and then the resultant 800 µL solution was transferred into a new tube. The solutions were incubated at room temperature for 5 minutes to allow complete dissociation of viral particles into the upper media and inactivation of any potentially remaining active virus in the solution. The RNA was then isolated from protein and DNA following the standard TRIzol phase separation procedure

Journal Pre-proof [48] . Precipitation of RNA was carried out by adding 1 mL 200-proof ethanol and 1 µL RNA-grade glycogen (R0551, ThermoFisher) to each tube, followed by 1-minute vortexing. Each tube was then incubated overnight at 20 C . Following overnight precipitation, the supernatant was discarded and residual ethanol was allowed to evaporate. The RNA pellet was washed following the standard TRIzol RNA wash procedure and subsequently re-suspended in 8 µL nuclease-free water.

Each 8 TRIzol isolation product was assayed with TaqPath 1-step RT-qPCR (A15299, ThermoFisher Scientific) in 20 µL reactions. We used probes from the CDC SARS-CoV-2 qPCR probe assay targeting two regions in the N gene, designated N1 and N2 (10006713, Integrated DNA Technologies), one for each sample replicate. To avoid cross-contamination, the reactions were loaded into non-adjacent wells in a 96-well plate on ice at a separate bench from where the RNA isolation step was performed. Wells also were covered with Parafilm between loading samples. RT-qPCR was carried out on a Quantstudio 3 (ThermoFisher) using the CDC-recommended protocol [49] .

October 14, 2021, filters were installed and collected in one day. In one-day testing, 0 filters returned positive, indicating that one day may not be enough filter exposure time to build up a detectable viral load. However, a relatively high rate (60%) of swab samples returned positive, which may be attributable to lack of surface decontamination mid-day. Metro cleans buses nightly, and the morning sampling period for all 2-week samples occurred the morning following the decontamination, in between which no riders would have ridden the bus. is not fully representative of the overall population from which individual testing was performed.

In control experiments, extracting spike control from filters yielded 6/9 (66.7%) replicates 

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