Development of a Duplex-ddPCR assay for accurate quantification of pseudorabies virus through systematic optimization of amplification bias

文献类型： 外文期刊

第一作者： Tian, Zihan

作者： Tian, Zihan;Li, Wentao;He, Qigai;Tian, Zihan;Li, Wentao;He, Qigai;Wu, Hao;Xu, Rong;Li, Wentao;He, Qigai;Yao, Lun

作者机构：

关键词： Droplet digital PCR; Pseudorabies virus; Absolute quantification; GC-Rich template

期刊名称：VIROLOGY （ 影响因子：2.4； 五年影响因子：2.5 ）

ISSN： 0042-6822

年卷期： 2025 年 602 卷

页码：

收录情况： SCI

摘要： Pseudorabies (PR), caused by the pseudorabies virus (PRV), is highly contagious. Although qPCR is widely used for viral DNA detection, it struggles with low-level DNA identification and precise quantification. To address these issues, droplet digital PCR (ddPCR) has emerged as a more advanced method for detecting pathogens and providing absolute quantification of nucleic acids. The study introduces a ddPCR assay for accurate PRV quantification, addressing the challenges posed by the high GC content of the PRV genome. By optimizing factors such as primer and probe concentrations, annealing conditions, denaturation time, and cycle number, the assay overcomes limitations of traditional PCR techniques. The optimized ddPCR assay showed a wide linear dynamic range, with well-defined limits of blank (LOB) and detection (LOD). Testing confirmed the method's reproducibility, demonstrating its stability and reliability. This study provides key insights into optimizing ddPCR for GC-rich templates and serves as a useful reference for future experiments.

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