Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics

Whale, Alexandra S.; Heide, Eva K. von der; Kohlenberg, Max; Brinckmann, Anja; Baedker, Silke; Fernández-González, Ana; Busby, Eloise; Bustin, Stephen A.; Hauser, Heiko; Missel, Andreas; O’Sullivan, Denise M; Huggett, Jim F.; Pfaffl, Michael W.; Nolan, Tania

doi:10.1016/j.ymeth.2021.08.006

Cited by 18 publications

(18 citation statements)

References 28 publications

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“…Limited yields of cells or fluids from sampling procedures such as nasopharyngeal swabs, and the presence of potential inhibitors (e.g., chemical or protein contaminants) in clinical samples, require that PCR amplification and detection be highly sensitive and reliable during SARS-CoV-2 nucleic acid analysis. Digital PCR has demonstrated significant advantages in both SARS-CoV-2 gRNA [94][95][96] and sgRNA [49,97,98] studies due to its ability for absolute quantification [98][99][100][101][102][103][104][105], tolerance to inhibitors [106], increased precision at low analyte copy numbers [107][108][109], and inter-run reproducibility [110][111][112]. One additional distinct advantage of the digital PCR approach is its lower susceptibility to sequence mismatches, which is especially relevant as emerging mutations that can potentially predominate could affect the performance of real-time PCR-based assays if they occur in regions where the PCR primer and probes are located [113,114].…”

Section: Analysis Approachesmentioning

confidence: 99%

SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives

Long

2021

Viruses

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SARS-CoV-2, the etiologic agent at the root of the ongoing COVID-19 pandemic, harbors a large RNA genome from which a tiered ensemble of subgenomic RNAs (sgRNAs) is generated. Comprehensive definition and investigation of these RNA products are important for understanding SARS-CoV-2 pathogenesis. This review summarizes the recent progress on SARS-CoV-2 sgRNA identification, characterization, and application as a viral replication marker. The significance of these findings and potential future research areas of interest are discussed.

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Section: Analysis Approachesmentioning

confidence: 99%

SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives

Long

2021

Viruses

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“… 34 − 38 The results from testing of clinical samples for SARS-CoV-2 RNA indicate RT-dPCR can be more accurate in diagnosing COVID-19 than RT-qPCR, and such results are promising for the application of dPCR in wastewater surveillance. 39 − 43 …”

Section: Introductionmentioning

confidence: 99%

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater

et al. 2022

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We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2–5 times lower than those using RT-qPCR. During sample processing, the endogenous ( n = 96) and exogenous ( n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

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“…Reference materials have played an important role in the molecular diagnosis of SARS-CoV-2 since the beginning of the pandemic. The establishment of stable, well-characterized reference materials helps not only in the validation of test results but also in the analysis of assays, workflows, devices, and SARS-CoV-2 diagnostic kits ( 70 , 71 , 79 – 81 ). Quantification by qPCR requires a sample of a known quantity to establish a standard curve, which in turn is used for relative quantification ( 27 , 30 , 72 ).…”

Section: Synoptic Overview Of Sars-cov-2 Rt-dpcr Applicationsmentioning

confidence: 99%