1887

Abstract

The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.

Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.

We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).

We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.

Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (=127; R=0.9259).

Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001301
2021-03-18
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jmm/70/3/jmm001301.html?itemId=/content/journal/jmm/10.1099/jmm.0.001301&mimeType=html&fmt=ahah

References

  1. Scally G, Jacobson B, Abbasi K. The UK's public health response to covid-19. BMJ 2020; 369:m1932 [View Article][PubMed]
    [Google Scholar]
  2. Andersson M, Low N, French N, Greenhalgh T, Jeffery K et al. Rapid roll out of SARS-CoV-2 antibody testing-a concern. BMJ 2020; 369:m2420 [View Article][PubMed]
    [Google Scholar]
  3. Ngo KAJ SA, Church TM. Unreliable inactivation of viruses by commonly used lysis buffers. Applied Biosafety 2017; 22:56–59
    [Google Scholar]
  4. Wang Y, Kang H, Liu X, Tong Z. Combination of RT-qPCR testing and clinical features for diagnosis of COVID-19 facilitates management of SARS-CoV-2 outbreak. J Med Virol 2020; 92:538–539 [View Article][PubMed]
    [Google Scholar]
  5. Kampf G, Voss A, Scheithauer S. Inactivation of coronaviruses by heat. J Hosp Infect 2020; 105:348–349 [View Article][PubMed]
    [Google Scholar]
  6. Darnell ME, Subbarao K, Feinstone SM, Taylor DR. Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV. J Virol Methods 2004; 121:85–91 [View Article][PubMed]
    [Google Scholar]
  7. Freire-Paspuel B, Vega-Marino PA, Velez A, Cruz M, Perez F. Evaluation of Viasure SARS-CoV-2 RT-qPCR kit (CerTest Biotec) using CDC FDA EUA RT-qPCR kit as a gold standard. medRxiv 2020 [View Article]
    [Google Scholar]
  8. Lista MJ, Page R, Sertkaya H, Matos P, Ortiz-Zapater E. Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation. medRxiv 2020 [View Article]
    [Google Scholar]
  9. Bosworth A, Whalley C, Poxon C, Wanigasooriya K, Pickles O et al. Rapid implementation and validation of a cold-chain free SARS-CoV-2 diagnostic testing workflow to support surge capacity. J Clin Virol 2020; 128:104469 [View Article][PubMed]
    [Google Scholar]
  10. Chan JF, Yip CC, To KK-W, Tang TH, Wong SC et al. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel Real-Time reverse Transcription-PCR assay validated in vitro and with clinical specimens. J Clin Microbiol 2020; 58: [View Article]
    [Google Scholar]
  11. Vogels CBF, Brito AF, Wyllie AL, Fauver JR, Ott IM et al. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets. Nat Microbiol 2020; 5:1299–1305 [View Article][PubMed]
    [Google Scholar]
  12. Vogels CBF, Watkins AE, Harden CA, Brackney D, Shafer J. SalivaDirect: a simplified and flexible platform to enhance SARS-CoV-2 testing capacity. medRxiv 2020 [View Article]
    [Google Scholar]
  13. Fomsgaard AS, Rosenstierne MW. An alternative workflow for molecular detection of SARS-CoV-2 – escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. Eurosurveillance 2020; 25:2000398 [View Article]
    [Google Scholar]
  14. Perchetti GA, Nalla AK, Huang ML, Jerome KR, Greninger AL. Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR. J Clin Virol 2020; 129:104499 [View Article]
    [Google Scholar]
  15. Waggoner JJ, Stittleburg V, Pond R, Saklawi Y, Sahoo MK et al. Triplex real-time RT-PCR for severe acute respiratory syndrome coronavirus 2. Emerg Infect Dis 2020; 26:1633–1635 [View Article]
    [Google Scholar]
  16. Larremore DB, Wilder B, Lester E, Shehata S, Burke JM et al. Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance. medRxiv 20202020.06.22.20136309 27 Jun 2020 [View Article][PubMed]
    [Google Scholar]
  17. Lavezzo E, Franchin E, Ciavarella C, Cuomo-Dannenburg G, Barzon L. Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo’. Nature 2020; 584:425–429 [View Article]
    [Google Scholar]
  18. Chau NVV, Lam T V, Thanh Dung N, Yen LM, Minh NNQ et al. The natural history and transmission potential of asymptomatic SARS-CoV-2 infection. Clin Infect Dis 2020
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.001301
Loading
/content/journal/jmm/10.1099/jmm.0.001301
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error