Molecular detection of SARS-CoV-2 using a reagent-free approach

Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Many groups recently presented results using heat processing method of respiratory...

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Bibliographic Details
Published in:PloS one 2020-12, Vol.15 (12), p.e0243266-e0243266
Main Authors: Calvez, Ronan, Taylor, Andrew, Calvo-Bado, Leonides, Fraser, Donald, Fink, Colin G
Format: Article
Language:English
Subjects:
Biology and life sciences
Clinical Laboratory Techniques - methods
Coronavirus Infections - epidemiology
Coronaviruses
COVID-19
COVID-19 - genetics
COVID-19 - metabolism
COVID-19 Testing - methods
Diagnosis
Disease transmission
Engineering and Technology
Genomes
Heat treatment
Heat treatments
Heating
Humans
Incubation
Indicators and Reagents
Laboratories
Medicine and health sciences
Methods
Molecular diagnostic techniques
Pandemics
Pneumonia, Viral - epidemiology
Reagents
Real-Time Polymerase Chain Reaction - methods
Research and Analysis Methods
Ribonucleic acid
RNA
RNA, Viral - genetics
Sample preparation
SARS-CoV-2 - genetics
SARS-CoV-2 - metabolism
Sensitivity and Specificity
Severe acute respiratory syndrome
Severe acute respiratory syndrome coronavirus 2
Specimen Handling - methods
Statistical analysis
Streamlining
Temperature
Transport media
Values
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Summary:Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Many groups recently presented results using heat processing method of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost. Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this methodology. We first investigated various treatments using different temperatures, incubation times and sample volumes to optimise the heat treatment conditions. Although the initial data confirmed results published elsewhere, further investigations revealed unexpected inhibitory properties of some commonly used universal transport media (UTMs) on some commercially available RT-qPCR mixes, leading to a risk of reporting false-negative results. This emphasises the critical importance of a thorough validation process to determine the most suitable reagents to use depending on the sample types to be tested. In conclusion, a heat processing method is effective with very consistent Ct values and a sensitivity of 96.2% when compared to a conventional RNA extraction method. It is also critical to include an internal control to check each sample for potential inhibition.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0243266