ABSTRACT
RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as "non-positive" (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , Color , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , France/epidemiology , Gene Expression , Humans , Limit of Detection , Nasopharynx/virology , Phosphoproteins/genetics , RNA Helicases/genetics , RNA-Dependent RNA Polymerase/genetics , Viral LoadABSTRACT
Cell culture medium, nasopharyngeal and sera samples spiked with SARS-CoV-2 were subjected to heat inactivation for various periods of time, ranging from 30 s to 60 min. Our results showed that SARS-CoV-2 could be inactivated in less than 30 min, 15 min, and 3 min at 56 °C, 65 °C, and 95 °C, respectively. These data could help laboratory workers to improve their protocols by handling the virus in biosafety conditions.