Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E

Nina M Pollak; Daniel J Rawle; Kexin Yan; Cameron Buckley; Thuy T Le; Claire Y T Wang; Nicole G Ertl; Karla van Huyssteen; Nicole Crkvencic; Misha Hashmi; Russell E Lyons; David M Whiley; Andreas Suhrbier; Joanne Macdonald

doi:10.3389/fmicb.2023.1238542

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E

Front Microbiol. 2023 Oct 6:14:1238542. doi: 10.3389/fmicb.2023.1238542. eCollection 2023.

Authors

Nina M Pollak^#^{1

2

3}, Daniel J Rawle^#⁴, Kexin Yan⁴, Cameron Buckley⁵, Thuy T Le⁴, Claire Y T Wang⁶, Nicole G Ertl⁵, Karla van Huyssteen⁷, Nicole Crkvencic⁸, Misha Hashmi⁸, Russell E Lyons⁷, David M Whiley^{5

9}, Andreas Suhrbier^{4

10}, Joanne Macdonald^{1

2

7}

Affiliations

¹ Center for Bioinnovation, University of the Sunshine Coast, Sippy Downs, QLD, Australia.
² School of Science, Technology and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, Australia.
³ DMTC Limited, Kew, VIC, Australia.
⁴ Inflammation Biology Group, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.
⁵ Faculty of Medicine, UQ Centre for Clinical Research, The University of Queensland, Herston, QLD, Australia.
⁶ Queensland Paediatric Infectious Diseases Laboratory, Centre for Children's Health Research, Brisbane, QLD, Australia.
⁷ BioCifer Pty Ltd., Auchenflower, QLD, Australia.
⁸ Bio Molecular Systems, Potts Point, NSW, Australia.
⁹ Microbiology Department, Pathology Queensland, Herston, QLD, Australia.
¹⁰ GVN Center of Excellence, Australian Infectious Disease Research Centre, Herston, QLD, Australia.

^# Contributed equally.

Abstract

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

Keywords: COVID-19; RNA extraction; RT-qPCR; SARS-CoV-2; diagnostics; safety; virus inactivation.

Grants and funding

This project was funded by DMTC Limited (Australia), the Medical Countermeasures Program (Project 10.75), and BioCifer Pty Ltd. Bio Molecular Systems (BMS) also contributed the instrumentation, proprietary master mix, and proprietary oligonucleotide mix, as well as staff time, during the testing on the BMS Mic and Myra instruments. This study was also supported, in part, by the Bill and Melinda Gates Foundation (OPP1140133). According to the Foundation's grant conditions, a Creative Commons Attribution 4.0 Generic License has already been assigned to the author's Accepted Manuscript version that may result from this submission. BioCifer, BMS, DMTC Limited, and the Bill and Melinda Gates Foundation had no involvement in the study design, collection, analysis, data interpretation, article writing, or the decision to submit it for publication. The establishment of the QIMR Berghofer MRI SARS-CoV-2/COVID-19 PC3 research facilities was supported by generous philanthropic donations from the Brazil Family Foundation (and others). Research within this PC3 facility for BioCifer was funded by R&D contracts from BioCifer. AS holds an Investigator grant from the National Health and Medical Research Council (NHMRC) of Australia (APP1173880).