Evaluation of droplet digital PCR for quantification of SARS-CoV-2 Virus in discharged COVID-19 patients

Liu, Chong; Shi, Qingxin; Peng, Minfei; Lu, Ruijie; Li, Haohao; Cai, Yingying; Chen, Jiaxi; Xu, Jiaqin; Shen, Bo

doi:10.18632/aging.104020

Cited by 35 publications

(34 citation statements)

References 14 publications

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“…Our finding that measurement using qPCR and ddPCR are highly correlated is consistent with other studies in nasopharyngeal swabs (Falzone et al 2020, Liu et al 2020), plasma (Tedim et al 2021), wastewater from aircraft and cruise ships (Ahmed et al 2020), and raw influent (Cieselski et al 2021). However, we observed that the correlation became weaker at the lower end of the concentration range we tested, spreading out at lower concentrations in a “broom-shaped” pattern consistent with studies comparing measurements of bacterial targets (Cao et al 2015).…”

Section: Discussionsupporting

confidence: 91%

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Steele

Zimmer‐Faust

Griffith

et al. 2021

Preprint

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The rapid emergence of wastewater based surveillance has led to a wide array of SARS-CoV-2 RNA quantification methodologies being employed. Here we compare methods to store samples, inactivate viruses, capture/concentrate viruses, and extract/measure viral RNA from primary influent into wastewater facilities. We found that heat inactivation of the viruses led to a 1-3 log10 decrease compared to chemical inactivation. Freezing influent prior to concentration caused a 1-4 log10 decrease compared to processing fresh samples, but viral capture by membrane adsorption prior to freezing was robust to freeze-thaw variability. Concentration vs. direct extraction, and PCR platform also affected outcome, but by a smaller amount. The choice of nucleocapsid gene target had nearly no effect. Pepper mild-mottle virus was much less sensitive to these methodological differences than was SARS-CoV-2, which challenges its use as a population-level control among studies using different methods. Better characterizing the variability associated with different methodologies, in particular the impact of methods on sensitivity, will aid decision makers in following the effects of vaccination campaigns, early detection of future outbreaks, and potentially monitoring the appearance of SARS-CoV-2 variants in the population.

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Section: Discussionsupporting

confidence: 91%

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Steele

Zimmer‐Faust

Griffith

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Conversely, ddPCR is able to accurately assess SARS-CoV-2 viral load, thus allowing clinicians to effectively diagnose COVID-19 infection, particularly in asymptomatic or paucisymptomatic patients ( 188 ). In addition, ddPCR may be used for the monitoring of COVID-19 patients, thus allowing the discharge of patients who have resolved the infection and to guarantee correct social isolation measures useful for limiting the spread of infections ( 189 ).…”

Section: Alternative Methods For the Effective Diagnosis Of Covid-mentioning

confidence: 99%

Current and innovative methods for the diagnosis of COVID‑19 infection (Review)

et al. 2021

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The Coronavirus Disease 2019 (COVID-19) pandemic has forced the scientific community to rapidly develop highly reliable diagnostic methods in order to effectively and accurately diagnose this pathology, thus limiting the spread of infection. Although the structural and molecular characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were initially unknown, various diagnostic strategies useful for making a correct diagnosis of COVID-19 have been rapidly developed by private research laboratories and biomedical companies. At present, rapid antigen or antibody tests, immunoenzymatic serological tests and molecular tests based on RT-PCR are the most widely used and validated techniques worldwide. Apart from these conventional methods, other techniques, including isothermal nucleic acid amplification techniques, clusters of regularly inter-spaced short palindromic repeats/Cas (CRISPR/Cas)-based approaches or digital PCR methods are currently used in research contexts or are awaiting approval for diagnostic use by competent authorities. In order to provide guidance for the correct use of COVID-19 diagnostic tests, the present review describes the diagnostic strategies available which may be used for the diagnosis of COVID-19 infection in both clinical and research settings. In particular, the technical and instrumental characteristics of the diagnostic methods used are described herein. In addition, updated and detailed information about the type of sample, the modality and the timing of use of specific tests are also discussed.

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“…In recent years, digital PCR (dPCR) has emerged as an attractive alternative for environmental applications as it offers absolute quantification without the need for a standard curve, and it may be more sensitive and less prone to inhibition (Cao et al, 2015). As applied to the detection of SARS-CoV-2, RT-dPCR has been reported to be superior to RT-qPCR for clinical specimens, including nasopharyngeal samples (Falzone et al, 2020;Liu et al, 2020;Suo et al, 2020) and plasma from infected patients (Tedim et al, 2021), as it was found to be more sensitive and accurate.…”

Section: Pcr Platformsmentioning

confidence: 99%

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Ahmed¹,

Simpson²,

Bertsch³

et al. 2021

Preprint

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Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.

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Evaluation of droplet digital PCR for quantification of SARS-CoV-2 Virus in discharged COVID-19 patients

Cited by 35 publications

References 14 publications

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Current and innovative methods for the diagnosis of COVID‑19 infection (Review)

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

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