Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR
| Autor: | Volkher Scharnhorst, Mieke Baselmans, Birgit Deiman, Remco de Kock |
|---|---|
| Přispěvatelé: | Chemical Biology, Eindhoven MedTech Innovation Center, EAISI Health |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Microbiology (medical) Monitoring viruses RT-PCR ddPCR Real-Time Polymerase Chain Reaction SDG 3 – Goede gezondheid en welzijn Autoantigens Sensitivity and Specificity Ribonuclease P 03 medical and health sciences Coronavirus Envelope Proteins 0302 clinical medicine SDG 3 - Good Health and Well-being Quantification Multiplex polymerase chain reaction Coronavirus Nucleocapsid Proteins Humans Multiplex RNA Messenger Glucuronidase Messenger RNA Coronavirus RNA-Dependent RNA Polymerase Genes Essential Chemistry Reverse Transcriptase Polymerase Chain Reaction SARS-CoV-2 RNA COVID-19 Assay sensitivity General Medicine DNA Phosphoproteins Molecular biology Housekeeping gene 030104 developmental biology Infectious Diseases 030220 oncology & carcinogenesis COVID-19 Nucleic Acid Testing Nucleic acid RNA Viral Original Article Viral load Multiplex Polymerase Chain Reaction |
| Zdroj: | European Journal of Clinical Microbiology & Infectious Diseases European Journal of Clinical Microbiology and Infectious Diseases, 40(4), 807-813. Springer |
| ISSN: | 1435-4373 0934-9723 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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