Environmental monitoring of SARS-CoV-2 for research and public health purposes has grown exponentially throughout the COVID-19 pandemic. Monitoring wastewater for SARS-CoV-2 provides early warning signals of virus spread and information on trends in infections at a community scale. Indoor environmental monitoring (e.g., swabbing of surfaces and air filters) to identify potential outbreaks is less common, and the evidence for its utility is mixed. A significant challenge with surface and air filter monitoring in this context is the concern of “relic RNA”— non-infectious RNA found in the environment that is not from a recently deposited virus. Here, we report the detection of SARS-CoV-2 RNA on surfaces in an isolation unit (a university dorm room) for up to eight months after a COVID-19-infected individual vacated the space. Comparison of sequencing results from the same location over two time points indicates the presence of the entire viral genome and sequence similarity confirms a single source of the virus. Our findings highlight the need to develop approaches that account for relic RNA in environmental monitoring.

Samples were collected using nylon fiber oral swabs with an ABS handle (Miraclean Technology Co. Ltd, China) that were pre-moistened in DNA/RNA Shield (Zymo Research, USA). An area of 10 cm x 10 cm (or equivalent) was swabbed for surface samples and a similar process was used to swab the exterior surface of the HEPA filter (~823 cm2) in a portable filtration unit. The same 10 cm x 10 cm area was swabbed every sampling time point until day 37. Afterward, a different 10 cm x 10 cm area was swabbed every time. RNA extraction and RT-qPCR were performed. Samples were extracted using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit (Applied Biosystems, USA) with a KingFisher Flex automated purification system (Thermo Fisher Scientific, USA). The MagMAX_Microbiome_Stool_Flex.bdz nucleic acid isolation protocol (Applied Biosystems, USA) was utilized, with modifications. In brief, the sample lysis step was not conducted as lysis was achieved through the use of DNA/RNA Shield and vortexing. All extracts were analyzed by RT-qPCR targeting the spike glycoprotein (S) gene of SARS-CoV-2 using the Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs Inc., USA).

Eight samples from this room were chosen for sequencing and combined with other samples from other related projects. The samples across projects were chosen to represent a variety of Ct values and surface types to calibrate future sequencing efforts. Ten μl of extracted nucleic acid from each of the 6 samples was converted to cDNA using the LunaScript RT SuperMix Kit (New England Biolabs, Ipswich, MA) in a 20 μl reaction mix, which was incubated at 25 °C for 2 min followed by 55 °C for 10 min and heat-inactivated at 95 °C for 1 min. Subsequently, 10 μl of this 1st-strand cDNA was used as input for amplification of the SARS-CoV-2 viral genome, using the xGen SARS-CoV-2 Amplicon Panel (IDT, Coralville, IA) which consist of 345 amplicons covering 99.7% of the SARS-CoV-2 Wuhan-Hu-1 strain (NC_045512.2). The workflow uses a single tube of tiled primer pairs resulting in an average amplicon size of 150 bp. The amplicon libraries were generated according to the workflow for low viral load workflows (Ct > 20) which involves two rounds of PCR rounds, a multiplex PCR (4 + 24 cycles), and the indexing PCR (5 cycles) to generate sequence-ready libraries. Libraries were barcoded with 8 bp unique dual indices during the indexing PCR. Equimolar libraries were pooled and quantified by qPCR with the KAPA Library Quantification Kits (Roche, Basel, Switzerland). The pooled library was sequenced on one lane of Illumina Mid Output NextSeq 500 (Illumina, San Diego, CA) with paired-end 150 bp reads. 

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