Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR
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ORIGINAL ARTICLE
Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR Remco de Kock 1,2,3
&
Mieke Baselmans 1 & Volkher Scharnhorst 1,2,3 & Birgit Deiman 1,2,3
Received: 15 September 2020 / Accepted: 14 October 2020 # The Author(s) 2020
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-CoV2 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. Keywords SARS-CoV-2 . ddPCR . Multiplex . RT-PCR . Quantification . Monitoring
Introduction The outbreak of Covid-19 caused by SARS-CoV-2 has spread worldwide. Up to now, over 35 million confirmed cases have been reported. The USA, Brazil, and India are the most affected countries with the highest mortality rates due to Covid-19 [1]. The gold standard for the detection of SARS-CoV-2 is based on real-time reverse transcriptase PCR (RT-PCR). In
* Remco de Kock [email protected] 1
Clinical Laboratory, Catharina Hospital Eindhoven, Eindhoven, The Netherlands
2
Institute for Complex Molecular Systems and Department of Biomedical Engineering, Laboratory of Chemical Biology, Eindhoven University of Technology, Eindhoven, The Netherlands
3
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]. In addition, to improve assay sensitivity, other
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