SARS-CoV-2 virus stability in GTR-VTM

Published: 28-09-2020| Version 2 | DOI: 10.17632/snz6rnyx3v.2
Contributor:
Shanavaz Nasarabadi

Description

GTR-VTM NP swab transport media was tested for stabilizing SARS-CoV-2 viral RNA and inactivated SARS-CoV-2 virus for up to 10 days at 4 temperatures. UniTranz-RT Transport System (UTM) was used to compare as this was the only viral transport media that we were able to source. Four temperatures were chosen; 3 are ambient temps and the 56C is a Department Of Transportation (DOT) suggested test temperature. Temperatures tested were 25C (refrigerated lab ambient temp), 37C (ambient), 46C (ambient) and 56C (transportation temp per DOT). Study setup: GTR-VTM and UTM samples were placed at the 4 temperatures after spiking with nasal matrix and either SARS-CoV-2 viral RNA (study 1) or virus (study 2). Matched control samples were placed at -80C. Analysis of samples was done following CDC's EUA approved protocol for COVID-19 RT-PCR molecular assay (https://www.fda.gov/media/134922/download). CDC's SARS-CoV-2 panel of oligonucleotides was sourced from Integrated DNA Technologies (IDT). The viral RNA was extracted from 140uL of sample with CDC suggested QIAamp viral RNA kit. RNA was eluted with 140uL of elution buffer. Five uL of extracted RNA sample was amplified with TaqPath master mix in a total volume of 20 uL per CDC's protocol. StepOne Plus thermal cycler was used for quantification. At each time point, -80C samples were extracted alongside experimental samples. Outcome: In study 1, (file: Stress Study 1- RNA spike-in) viral RNA spiked into GTR-VTM was stable for 10 days at 25C and 37C and 5 days at 46C and 56C. No RNA was recovered from SARS-CoV-2 viral RNA spiked in UTM as the RNA degraded immediately, so, UTM samples with spiked RNA were abandoned in study 1 after day 1. The only way to recover RNA from UTM was to add lysis buffer first to the UTM and then spike with viral RNA (data not shown). Percent recovery of RNA was determined with comparison to the matched -80C controls. Each sheet represents the RT-PCR data for a single temperatures and day e.g. Study 1_25C, d1 means Day 1 collected samples for spiked viral RNA in GTR-VTM incubated at 25C, and so on. The -80C controls for all days are from rows G1 to H12. Only the 1/125 samples for the GTR-VTM and UTM data have been reported in the manuscript. ANOVA analysis of study 2 data, (file: Study 2 - Virus spike in ANOVA) performed by Summer Rose. A rejection threshold of (CTCtrl + 3) was weighed against each sample based on the -80C control values. ANOVA predicts that GTR-VTM will stabilize the SARS-CoV-2 virus at all temperatures for up to 23 days at all 4 temperatures. SARS-CoV-2 virus spiked in UTM will stabilize virus for 10 days and 5 days at 25C and 37C resp., but not at 46C and 56C. GTR-VTM chemistry inhibits microbial growth and RNase activity to keep the viral RNA and virus stable for extended periods of time (VTM 65C and 70C Heat Test.xlsx) at high temperature. Quantity of RNA from GTR-VTM (Magnetic-beads-Experiment.xlsx) NP samples is similar for both QuickRNA and MagMax kits.

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