A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

Wagner, Kaitlyn; Fox, Philip D.; Gordon, Elizabeth; Hahn, Westen; Olsen, Kenzie; Markham, Alex; Buglewicz, Dylan J.; Selemenakis, Platon; Lessard, Avery; Goldstein, Daniella; Threatt, Alissa; Davis, Luke; Miller-Dawson, Jake; Stockett, Halie; Rugh, Kristin; Turner, Houston; Remias, Michelle; Williams, Maggie; Chavez, Jorge; Galindo, Gabriel; Cialek, Charlotte; Koch, Amanda; Fout, Alex; Fosdick, Bailey K.; Broeckling, Bettina E.; Zabel, Mark D.

doi:10.1038/s41598-023-29858-5

Cited by 3 publications

(4 citation statements)

References 59 publications

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“…However, pooling samples can be challenging, and various factors can influence the sensitivity of the screening procedure. Studies have shown that sensitivity decreases with increased pool size 29 . In addition, the efficiency of the pooled tests has been reported to depend on the viral load of the samples, the dilution of the sample 30 and also the transportation mode, storage and sensitivity of the kit 31 .…”

Section: Discussionmentioning

confidence: 99%

“…Fibre swabs are bound in layers and are not conducive to scraping epithelial cells and releasing viruses 30 . Flocked swabs are made by an electrostatic flocking process that attaches millions of nylon microfibers vertically to the swab head, facilitating sample collection, keeping the sample on the swab surface, and allowing for easier virus elution 29 .…”

Section: Discussionmentioning

confidence: 99%

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Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR

Zhang,

Shen,

Liang

et al. 2024

Sci Rep

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Though pooling samples for SARS-CoV-2 detection has effectively met the need for rapid diagnostic and screening tests, many factors can influence the sensitivity of a pooled test. In this study, we conducted a simulation experiment to evaluate modes of pooling specimens and aimed at formulating an optimal pooling strategy. We focussed on the type of swab, their solvent adsorption ability, pool size, pooling volume, and different factors affecting the quality of preserving RNA by different virus solutions. Both quantitative PCR and digital PCR were used to evaluate the sampling performance. In addition, we determined the detection limit by sampling which is simulated from the virus of different titers and evaluated the effect of sample-storage conditions by determining the viral load after storage. We found that flocked swabs were better than fibre swabs. The RNA-preserving ability of the non-inactivating virus solution was slightly better than that of the inactivating virus solution. The optimal pooling strategy was a pool size of 10 samples in a total volume of 9 mL. Storing the collected samples at 4 °C or 25 °C for up to 48 h had little effect on the detection sensitivity. Further, we observed that our optimal pooling strategy performed equally well as the single-tube test did. In clinical applications, we recommend adopting this pooling strategy for low-risk populations to improve screening efficiency and shape future strategies for detecting and managing other respiratory pathogens, thus contributing to preparedness for future public health challenges.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR

Zhang,

Shen,

Liang

et al. 2024

Sci Rep

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“…Studies have shown RT-dPCR assays to be efficient in different samples like saliva, plasma, formalin-fixed paraffin-embedded (FFPE) tissue etc. [109][110][111] However, efforts should be made to ensure the performance characteristics of the developed dPCR assays in different sample types.…”

Section: Development and Validation Of Diagnostic Methods And Kitsmentioning

confidence: 99%

“…Furthermore, these analytical validation guidelines are compatible with different starting materials/samples like saliva, tissue, blood etc. Studies have shown RT‐dPCR assays to be efficient in different samples like saliva, plasma, formalin‐fixed paraffin‐embedded (FFPE) tissue etc 109–111 . However, efforts should be made to ensure the performance characteristics of the developed dPCR assays in different sample types.…”

Section: Applications Of Digital Pcr In Sars‐cov‐2 Quantificationmentioning

confidence: 99%

SARS‐CoV‐2 in digital era: Diagnostic techniques and importance of nucleic acid quantification with digital PCRs

Aravind Kumar,

Aradhana,

Harleen

et al. 2023

Reviews in Medical Virology

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Studies related to clinical diagnosis and research of SARS‐CoV‐2 are important in the current pandemic era. Although molecular biology has emphasised the importance of qualitative analysis, quantitative analysis with nucleic acids in relation to SARS‐CoV‐2 needs to be clearly emphasised, which can provide perspective for viral dynamic studies of SARS‐CoV‐2. In this regard, the requirement and utilization of digital PCR in COVID‐19 research has substantially increased during the pandemic, necessitating the aggregation of its cardinal applications and future scopes. Hence, this meta‐review comprehensively addresses and emphasises the importance of nucleic acid quantification of SARS‐CoV‐2 RNA with digital PCR (dPCR). Various quantitative techniques of clinical significance like immunological, proteomic and nucleic acid‐based diagnosis and quantification, have been comparatively discussed. Furthermore, the core part of the article focusses on the working principle and advantages of digital PCR, along with its applications in COVID‐19 research. Several important applications like viral load quantitation, environmental surveillance and assay validation have been extensively investigated and discussed. Certain key future scopes of clinical importance, like mortality prediction, viral/variant‐symbiosis, and antiviral studies were also identified, suggesting several possible digital PCR applications in COVID‐19 research.

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Optimization and application of digital droplet PCR for the detection of SARS-CoV-2 in saliva specimen using commercially available kit

Kaisar,

Kristin,

Wijaya

et al. 2024

Biology Methods and Protocols

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The coronavirus disease-19 (COVID-19) pandemic has resulted in a significant global health crisis, causing hundreds of millions of cases and millions of deaths. Despite being declared endemic, SARS-CoV-2 infection continues to pose a significant risk, particularly for immunocompromised individuals, highlighting the need for a more sensitive and specific detection. Reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) possesses a sensitive and absolute quantification compared to the gold standard. This study is the first to optimize RT-ddPCR for detecting SARS-CoV-2 in saliva specimens using a commercially available RT-qPCR kit. Optimization involved the assessment of the RT-ddPCR reaction mixture, annealing temperature adjustments, and validation using 40 stored saliva specimens. RT-qPCR was used as a reference method in this study. Compatibility assessment revealed that ddPCR Supermix for Probes (no dUTP) was preferable with an optimal annealing temperature of 57.6 °C. Although a 25% higher primer/probe concentration provides a higher amplitude in droplet separation of positive control, the number of copy numbers decreased. An inverse correlation between Ct value and copy number concentration was displayed, presenting that the lower the Ct value, the higher the concentration, for the N and E genes with r2 values of 0.98 and 0.85, respectively. However, ORF1ab was poorly correlated (r2 of 0.34). The sensitivity of targeted and E genes were 100% and 93.3%; as for the specificity, the percentage ranged from 80.8% to 91.3%. This study implicates the applicability of a modified method in the ddPCR platform for similar types of pathogens using saliva specimens.

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A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

Cited by 3 publications

References 59 publications

Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR

Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR

SARS‐CoV‐2 in digital era: Diagnostic techniques and importance of nucleic acid quantification with digital PCRs

Optimization and application of digital droplet PCR for the detection of SARS-CoV-2 in saliva specimen using commercially available kit

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