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

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

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Article number104416
Pages (from-to)1-5
Number of pages5
JournalJournal of Clinical Virology
Publication statusPublished - Jul 2020


  • Rapid response
  • Real-time RT-PCR
  • SARS-CoV-2
  • Surveillance

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases


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