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
作者： Tian, Zihan 1 ; Wu, Hao 3 ; Xu, Rong 4 ; Yao, Lun 5 ; Li, Wentao 1 ; He, Qigai 1 ;
作者机构： 1.Huazhong Agr Univ, Coll Vet Med, Natl Key Lab Agr Microbiol, Wuhan 430070, Peoples R China
2.Huazhong Agr Univ, Cooperat Innovat Ctr Sustainable Pig Prod, Wuhan 430070, Peoples R China
3.Guizhou Acad Agr Sci, Inst Anim Husb & Vet Sci, Guiyang 550005, Guizhou, Peoples R China
4.Huazhong Agr Univ, Anim Dis Diagnost Ctr, Wuhan 430070, Peoples R China
5.Hubei Acad Agr Sci, Inst Anim Husb & Vet Sci, Wuhan 430064, Peoples R China
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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