Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management

Viruses

Fabio Gentilini¹  Agata Scalcione²  Silvia Zannoli²  MariaMichela Marino²  Laura Grumiro²  Giorgio Dirani²  Martina Brandolini²  Francesca Taddei²  Vittorio Sambri²  Michela Fantini²  MariaElena Turba³ 

[1] Department of Veterinary Medical Sciences, Alma Mater Studiorum—University of Bologna, 40064 Bologna, Italy;Unit of Microbiology, The Great Romagna Hub Laboratory, 47522 Pievesestina, Italy;Xenturion Srl, 47122 Forlì, Italy;

关键词: SARS-CoV-2;    COVID-19;    qRT-PCR;    Ct;    dPCR;    TCID50/mL;   

DOI  :  10.3390/v13061022

来源: DOAJ

【 摘 要 】

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.

【 授权许可】

Unknown