ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Infectious Agents and Disease
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1635733
This article is part of the Research TopicPathogenic microorganisms and biosafetyView all 9 articles
Droplet digital RT-PCR method for SARS-CoV-2 variants detection in clinical and wastewater samples
Provisionally accepted
- Zhejiang Key Laboratory of Public Health Detection and Pathogenesis Research, Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention (Zhejiang CDC), Hangzhou, China
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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.Conclusions: 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.
Keywords: Droplet digital RT-PCR, SARS-COV-2 variants, clinical, wastewater, Sensitivity, specificity, repeatability
Received: 27 May 2025; Accepted: 18 Jun 2025.
Copyright: © 2025 Wang, Sun, Gong, Su, Zhou, Lou, Chen, Shi, Mao and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Haiyan Mao, Zhejiang Key Laboratory of Public Health Detection and Pathogenesis Research, Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention (Zhejiang CDC), Hangzhou, China
Yanjun Zhang, Zhejiang Key Laboratory of Public Health Detection and Pathogenesis Research, Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention (Zhejiang CDC), Hangzhou, China
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