Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation
Authors: Maria Jose Lista et al.
Journal/ Pre-Print: Medrxiv
Tags: Clinical, Diagnostics
1. Testing of three different RNA extraction kits, and three different one-step RT-qPCR
kits achieved almost comparable detection of viral RNA within patient swab material.
2. Different recommended primer probe sets (N1, N2, RdRP) were assessed and the N1 and N2 primer-probes are more sensitive than RdRP.
3. Heat inactivation of virus within swab-material does not affect viral RNA detection and may be used to reduce the need for CL-3 laboratories.
In order to increase the SARS-CoV-2 testing capacity despite the shortage of reagents, the use of alternative strategies is required. Two viral RNA purification kits (Beckman and Omega Bio-tek) were tested as alternatives to the standard QIAGEN kit. All kits recover similar amounts of viral RNA from clinical swab material. The use of different RT-qPCR Master Mix brands (Fast Virus, PCR Biosystems and Luna) were compared, with FastVirus and Luna showing better results. Different primer probe sets (N1, N2, RdRP) were analysed and heat inactivation of swabs was discovered as a suitable method that does not affect viral RNA detection but makes is possible outside of CL-3 facilities. Lab SOP resource at https://osf.io/uebvj/.
Impact for SARS-CoV2/COVID19 research efforts
Develop diagnostic tools for SARS-CoV2/COVID19
(study establishes several testing protocols with different reagents, which can be implemented in labs)
· In vitro study
· Clinical Cohort study
Strengths and limitations of the paper
Novelty: The paper does not study novel techniques, but rather validates the reagents used in currently available methods and combines them in a pipeline.
Standing in the field: The paper uses and analyses well established methods for RNA extraction and RT-qPCR in the field. The thermal inactivation of the virus is controversially discussed in literature. Effective heat inactivation of SARS-CoV-2 was shown (e.g. by Chin A et al., doi:10.1101/2020.03.15.20036673), however, others claim that it affects the efficiency of RT-PCR for SARS-CoV-2 detection (Pan Y et al, American Association for Clinical Chemistry 2020).
Appropriate statistics: Due to small sample size not a lot of statistical analysis was performed. Only the correlation coefficient R was (appropriately) calculated.
Viral model used: SARS-CoV-2 clinical swab material from St Thomas’ Hospital (London, UK)
Translatability: As there is a high demand for more COVID-19 testing, but a shortage in reagents, the use of available alternatives has high translatability. The NHS agreed on the use of validated alternative RNA isolation and qPCR protocols, which increases the potential for quick implementation in labs.
Main limitations: The sample size is relatively small (4-10 clinical samples), which makes it difficult to get highly reliable results.
Some (3/10) negative control samples tested borderline positive/negative with heat inactivation by duplicate RT-qPCR, but this was not discussed.