Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

Abstract Purpose. We aimed to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive detection of SARS-CoV-2 RNA with respect to human derived RNA and could be used for triage and monitoring of Covid-19 patients. Methods. 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. Results. Assay sensitivity of the RT-PCR assay drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared to water, while the sensitivity of the RT-ddPCR was not affected by the total NA background. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality. 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. Conclusion. The present study describes a robust and sensitive one-step RT-ddPCR multiplex assay for reliable detection and 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.

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Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-020-04076-3 ◽

2020 ◽

Author(s):

Remco de Kock ◽

Mieke Baselmans ◽

Volkher Scharnhorst ◽

Birgit Deiman

Keyword(s):

Simultaneous Detection ◽

Housekeeping Gene ◽

Multiplex Assay ◽

Viral Rna ◽

Rt Pcr ◽

Pcr Assay ◽

Assay Sensitivity ◽

Viral Loads ◽

Pcr Multiplex ◽

One Step

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.

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