Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification
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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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SciScore for 10.1101/2020.09.02.20186023: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Human nasopharyngeal swab sample collection and preparation from COVID positive patients: Clinical nasopharyngeal swab samples from patients infected with SARS-CoV-2 were collected in UTM and acquired by the Chiu laboratory with approval of the University of California San Francisco (UCSF) Institutional Review Board (IRB).
Consent: The approved study was a no-subject contact biobanking protocol using remnant clinical samples with waiver of consent under approval from the University of California San Francisco (UCSF) Institutional Review Board (IRB).Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not … SciScore for 10.1101/2020.09.02.20186023: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Human nasopharyngeal swab sample collection and preparation from COVID positive patients: Clinical nasopharyngeal swab samples from patients infected with SARS-CoV-2 were collected in UTM and acquired by the Chiu laboratory with approval of the University of California San Francisco (UCSF) Institutional Review Board (IRB).
Consent: The approved study was a no-subject contact biobanking protocol using remnant clinical samples with waiver of consent under approval from the University of California San Francisco (UCSF) Institutional Review Board (IRB).Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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