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Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification.
Vasudevan, Harish N; Xu, Peng; Servellita, Venice; Miller, Steve; Liu, Leqian; Gopez, Allan; Chiu, Charles Y; Abate, Adam R.
  • Vasudevan HN; Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1700 4th St, San Francisco, CA, USA.
  • Xu P; Department of Radiation Oncology, University of California San Francisco, San Francisco, CA, USA.
  • Servellita V; Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1700 4th St, San Francisco, CA, USA.
  • Miller S; Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA.
  • Liu L; UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, USA.
  • Gopez A; Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA.
  • Chiu CY; Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1700 4th St, San Francisco, CA, USA.
  • Abate AR; Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA.
Sci Rep ; 11(1): 780, 2021 01 12.
Article in English | MEDLINE | ID: covidwho-1026832
Preprint
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ABSTRACT
The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Viral Load / COVID-19 Nucleic Acid Testing / COVID-19 Type of study: Diagnostic study / Prognostic study Topics: Long Covid Limits: Humans Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-020-80715-1

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Viral Load / COVID-19 Nucleic Acid Testing / COVID-19 Type of study: Diagnostic study / Prognostic study Topics: Long Covid Limits: Humans Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-020-80715-1