ArticleFrontiers in microbiology2025
Droplet digital RT-PCR method for SARS-CoV-2 variants detection in clinical and wastewater samples.
Article in Frontiers in microbiology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers, 1 of them a synthesis that pooled it.
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Who cites it
5 citing papers in PubMed, 1 synthesis or guideline pooled it.
- Methodological Approaches to Dengue Virus Detection in Wastewater: A Systematic Review and Meta-Analysis of Positivity Rate.Viruses · 2026Pooled it
- ACE2-PNA conjugates exploit viral endocytosis for targeted intracellular delivery and exhibit dual antiviral efficacy against SARS-CoV-2.Molecular biomedicine · 2026Article
- Diagnostic Performance of Droplet Digital PCR for Detecting Pathogens and Resistance Genes in Patients with Suspected Bloodstream Infections.Infection and drug resistance · 2026Article
- Integrating isothermal amplification with magnetic flocculation and DNA-guided PfAgo for SARS-CoV-2 detection.Frontiers in microbiology · 2026Article
- One Assay, Nine Targets: Advancing Viral Surveillance with Multiplex RT-ddPCR.Analytical chemistry · 2025Article
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10 authors.
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Abstract
Objective: To establish a sensitive, specific, and precise quantitative detection method for SARS-CoV-2 variants using droplet digital RT-PCR (RT-ddPCR). Methods: Dual primer-probe sets targeting the SARS-CoV-2 nucleocapsid (N) and spike (S) genes were designed. The annealing temperature for RT-ddPCR was optimized using a gradient PCR system. The sensitivity, defined as the limit of detection (LOD), was determined by serially diluting SARS-CoV-2 RNA. The specificity of the RT-ddPCR assay was evaluated using SARS-CoV-2 variants and common respiratory viruses. Precision and repeatability were assessed by quantitatively repeating the detection on serial dilutions of SARS-CoV-2 RNA. Additionally, the results of RT-ddPCR for clinical and environmental wastewater samples were compared with those from RT-qPCR. Results: The optimal annealing temperature was 53.5°C. The LOD for the N and S genes of the original SARS-CoV-2 strain was 4.26 (95% CI: 3.12-9.89) and 3.87 (95% CI: 2.77-7.75) copies/reaction. The Delta strain exhibited LODs of 4.65 (N gene, 95% CI: 3.28-9.64) and 6.12 (S gene, 95% CI: 4.33-15.59) copies/reaction. The Omicron showed 4.07 (N gene, 95% CI: 3.11-6.26) and 4.58 (S gene, 95% CI: 3.43-7.40) copies/reaction. Importantly, the RT-ddPCR assay was repeatable with a coefficient of variation of less than 10% when RNA concentrations of SARS-CoV-2 were between 73.50 and 7,500 copies/reaction. The high specificity of the RT-ddPCR assay was demonstrated by its ability to correctly detect the thirty SARS-CoV-2 variants, while not other common respiratory viruses. For 148 clinical pharyngeal swab specimens, the positive rate for both RT-ddPCR and RT-qPCR was 86.49%, and a coincidence rate of 98.65% and a Kappa value of 0.94. Quantitative comparison of RT-ddPCR and RT-qPCR in 50 wastewater samples with low viral load, RT-ddPCR assay detected 50 positives for dual gene targets (N and S genes), whereas RT-qPCR assay only 21 exhibited concurrent positivity for dual gene targets, while 25 showed S gene detection, and 4 were negative for dual gene targets, suggesting our RT-ddPCR assay enabled absolute quantification of SARS-CoV-2 variants with low viral load. Conclusion: The RT-ddPCR assay developed in this study can be used for SARS-CoV-2 variants detection and quantitative analysis of clinical and environmental samples.
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