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One-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification.
Nyaruaba, Raphael; Li, Xiohong; Mwaliko, Caroline; Ogolla, Faith; Li, Changchang; Zhao, Lu; Yang, Hang; Yu, Junping; Wei, Honping.
  • Nyaruaba R; Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
  • Li X; International College, University of Chinese Academy of Sciences, Beijing, China.
  • Mwaliko C; Sino-Africa Joint Research Center, Nairobi, Kenya.
  • Ogolla F; Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
  • Li C; International College, University of Chinese Academy of Sciences, Beijing, China.
  • Zhao L; Sino-Africa Joint Research Center, Nairobi, Kenya.
  • Yang H; CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Wuhan, Hubei, China.
  • Yu J; Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
  • Wei H; International College, University of Chinese Academy of Sciences, Beijing, China.
Methods Mol Biol ; 2452: 147-166, 2022.
Article in English | MEDLINE | ID: covidwho-1844265
ABSTRACT
Droplet digital polymerase chain reaction (ddPCR) is a third generation of PCR that was recently developed to overcome the limitation of direct quantification observed in real-time quantification PCR (qPCR). Recent studies have shown that ddPCR is more sensitive than the gold standard reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples. In combination with multiplexing, multiple RT-ddPCR assays can be developed to directly quantify different SARS-CoV-2 nucleic acid targets within a single sample, significantly saving on cost and time. Since ddPCR is tolerant to a number of inhibitors unlike qPCR, it can be used to detect and quantify samples from complex environments like wastewater. Here we present three one-step RT-ddPCR protocols on how to develop simplex (one target), duplex (two targets), and triplex probe mix (three targets) assays for SARS-CoV-2 detection and quantification. The assays can be used for diagnosis or other research-related SARS-CoV-2 applications.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study Limits: Humans Language: English Journal: Methods Mol Biol Journal subject: Molecular Biology Year: 2022 Document Type: Article Affiliation country: 978-1-0716-2111-0_10

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study Limits: Humans Language: English Journal: Methods Mol Biol Journal subject: Molecular Biology Year: 2022 Document Type: Article Affiliation country: 978-1-0716-2111-0_10