DOI: 10.25338/b8gp94
title: SARS-CoV-2 RNA detectable at least eight months after shedding in an isolation facility

description: 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.
description: 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
 &gt; 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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