Abstract
The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.
Highlights
The outbreak of Covid-19 caused by SARS-CoV-2 has spread worldwide
The goal of this study is to develop a sensitive one-step droplet digital reverse transcriptase PCR (RT-PCR) (RT-droplet digital polymerase chain reaction (ddPCR)) multiplex assay for simultaneous detection of multiple SARS-CoV-2 genes N (N1+ N2), E, and RNA-dependent RNA polymerase (RdRp), including the detection of patient-derived mRNA of a housekeeping gene to assure sample and assay quality and to enable quantification of viral RNA
The assay was performed in a 10 μL reaction volume, consisting of 5 μL 2× reaction buffer, 0.16 μL of a 50 mM magnesium sulfate solution and 0.40 μL of SuperScriptTM III RT/PlatinumTM Taq Mix, 400 nM forward primer, 400 nM reverse primer, 200 nM probe, 0.4 μg of nonacetylated bovine serum albumin (UltrapureTM BSA, Invitrogen, USA), and 2 μL of RNA
Summary
The outbreak of Covid-19 caused by SARS-CoV-2 has spread worldwide. The gold standard for the detection of SARS-CoV-2 is based on real-time reverse transcriptase PCR (RT-PCR). Expert Center Clinical Chemistry Eindhoven, Eindhoven, The Netherlands the Netherlands, the detection of the envelope (E) gene, followed by confirmatory testing of the RNA-dependent RNA polymerase (RdRp) gene, is recommended [2]. Another approach is to detect the nucleocapsid (N) gene and to use an open reading frame 1a/b (ORF1b) gene or E gene assay as a confirmatory test [3]. To improve assay sensitivity, other studies have been focusing on the detection of N, E, or ORF1b using droplet digital polymerase chain reaction (ddPCR) [4,5,6,7]
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