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. 2020 Jul:128:104416.
doi: 10.1016/j.jcv.2020.104416. Epub 2020 May 4.

Rapid and sensitive detection of SARS-CoV-2 RNA using the Simplexa™ COVID-19 direct assay

Licia Bordi  1 , Antonio Piralla  2 , Eleonora Lalle  1 , Federica Giardina  2 , Francesca Colavita  1 , Monica Tallarita  2 , Giuseppe Sberna  1 , Federica Novazzi  2 , Silvia Meschi  1 , Concetta Castilletti  1 , Angela Brisci  3 , Giulia Minnucci  3 , Veronica Tettamanzi  3 , Fausto Baldanti  4 , Maria Rosaria Capobianchi  1
Affiliations

Rapid and sensitive detection of SARS-CoV-2 RNA using the Simplexa™ COVID-19 direct assay

Licia Bordi et al. J Clin Virol. 2020 Jul.

Abstract

Background: So far, one of the major drawbacks of the available molecular assays for the diagnosis of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) is the need for viral nucleic acid extraction from clinical specimens.

Objective: The aim of this study was to evaluate the performances of a newly designed real-time RT-PCR (Simplexa™ COVID-19 Direct assay), that is established with an all-in-one reagent mix and no separate extraction required.

Results: The lower limit of detection (LOD) for both target genes resulted the same: 3.2 (CI: 2.9-3.8) log10 cp/mL and 0.40 (CI: 0.2-1.5) TCID50/mL for S gene while 3.2 log10 (CI: 2.9-3.7) log10 cp/mL and 0.4 (CI: 0.2-1.3) TCID50/mL for ORF1ab. The LOD obtained with extracted viral RNA for both S gene or ORF1ab was 2.7 log10 cp/mL. Crossreactive analysis performed in 20 nasopharyngeal swabs confirmed a 100% of clinical specificity of the assay. Clinical performances of Simplexa™ COVID-19 Direct assay were assessed in 278 nasopharyngeal swabs tested in parallel with Corman's method. Concordance analysis showed an "almost perfect" agreement in SARS-CoV-2 RNA detection between the two assays, being κ = 0.938; SE = 0.021; 95% CI = 0.896-0.980.

Conclusions: The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, enabling highspeed detection in just over one hour, which is significantly faster than the up to five hours currently required by traditional extraction followed by amplification technologies, thus allowing prompt decision making regarding isolation of infected patients.

Keywords: Rapid response; Real-time RT-PCR; SARS-CoV-2; Surveillance.

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Conflict of interest statement

Declaration of Competing Interest Angela Brisci, Giulia Minnucci, Veronica Tettamanzi are employees of the DiaSorin S.p.A. (Gerenzano, Italy). In no way their contribution to the completion of the study influenced the study design and the analysis of the results. No additional conflict of interest or other competing relationships exist.

Figures

Fig. 1
Fig. 1
Probit analysis for Simplexa™ COVID-19 Direct assay.
See this image and copyright information in PMC

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