Wang Feng, Sun Yi, Gong Liming, Su Lingxuan, Zhou Biaofeng, Lou Xiuyu, Chen Yin, Shi Wen, Mao Haiyan, Zhang Yanjun
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.
Front Microbiol. 2025 Jul 3;16:1635733. doi: 10.3389/fmicb.2025.1635733. eCollection 2025.
To establish a sensitive, specific, and precise quantitative detection method for SARS-CoV-2 variants using droplet digital RT-PCR (RT-ddPCR).
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.
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.
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.
建立一种使用液滴数字逆转录聚合酶链反应（RT-ddPCR）对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）变异株进行灵敏、特异且精确的定量检测方法。
设计针对SARS-CoV-2核衣壳（N）基因和刺突（S）基因的双引物-探针组。使用梯度PCR系统优化RT-ddPCR的退火温度。通过对SARS-CoV-2 RNA进行系列稀释来确定灵敏度，即检测限（LOD）。使用SARS-CoV-2变异株和常见呼吸道病毒评估RT-ddPCR检测的特异性。通过对SARS-CoV-2 RNA系列稀释液进行定量重复检测来评估精密度和重复性。此外，将临床和环境废水样本的RT-ddPCR结果与逆转录定量聚合酶链反应（RT-qPCR）结果进行比较。
最佳退火温度为53.5°C。原始SARS-CoV-2毒株N基因和S基因的LOD分别为4.26（95%置信区间：3.12 - 9.89）和3.87（95%置信区间：2.77 - 7.75）拷贝/反应。德尔塔毒株N基因和S基因的LOD分别为4.65（95%置信区间：3.28 - 9.64）和6.12（95%置信区间：4.33 - 15.59）拷贝/反应。奥密克戎毒株N基因和S基因的LOD分别为4.07（95%置信区间：3.11 - 6.26）和4.58（95%置信区间：3.43 - 7.40）拷贝/反应。重要的是，当SARS-CoV-2的RNA浓度在73.50至7500拷贝/反应之间时，RT-ddPCR检测具有可重复性，变异系数小于10%。RT-ddPCR检测的高特异性体现在其能够正确检测30种SARS-CoV-2变异株，而不会检测其他常见呼吸道病毒。对于148份临床咽拭子标本，RT-ddPCR和RT-qPCR的阳性率均为86.49%，符合率为98.65%，kappa值为0.94。在50份低病毒载量的废水样本中进行RT-ddPCR和RT-qPCR的定量比较，RT-ddPCR检测到双基因靶点（N基因和S基因）50份阳性，而RT-qPCR检测只有21份双基因靶点同时呈阳性，25份S基因检测呈阳性，4份双基因靶点呈阴性，表明我们的RT-ddPCR检测能够对低病毒载量的SARS-CoV-2变异株进行绝对定量。
本研究开发的RT-ddPCR检测方法可用于SARS-CoV-2变异株检测以及临床和环境样本的定量分析。
