Jelenčič Amadej, Dobnik David, Bogožalec Košir Alexandra, Mehle Nataša
Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.
Josef Stefan International Postgraduate School, Ljubljana, Slovenia.
Methods Mol Biol. 2025;2943:31-45. doi: 10.1007/978-1-0716-4642-7_3.
Pepino mosaic virus (PepMV) is a plant pathogen causing significant economic losses in tomato production. Sensitive, reliable, and robust detection methods are crucial for containing the spread of PepMV and reducing its damaging effects. Digital PCR (dPCR) presents several advantages to conventional real-time quantitative PCR (qPCR), including absolute quantification ability, robust quantitative multiplexing capabilities, and straightforward result analysis. Furthermore, dPCR is especially suitable for analysis of complex samples due to its remarkable tolerance to PCR inhibitors, which makes it a promising method for plant virus genotyping. In this chapter, we present two protocols for PepMV genotyping and quantification using one-step reverse transcription digital PCR (RT-dPCR). The first protocol outlines four simplex assays, while the second describes two duplex assays for precise and comprehensive genotyping of PepMV variants.
番茄潜叶蛾花叶病毒（PepMV）是一种植物病原体，在番茄生产中造成重大经济损失。灵敏、可靠且稳健的检测方法对于遏制PepMV的传播和减轻其破坏作用至关重要。数字PCR（dPCR）相较于传统实时定量PCR（qPCR）具有多个优势，包括绝对定量能力、强大的定量多重分析能力以及直接的结果分析。此外，dPCR因其对PCR抑制剂具有显著耐受性，特别适用于复杂样品的分析，这使其成为植物病毒基因分型的一种有前景的方法。在本章中，我们介绍了两种使用一步法逆转录数字PCR（RT-dPCR）对PepMV进行基因分型和定量的方案。第一个方案概述了四种单重分析方法，而第二个方案描述了两种双重分析方法，用于对PepMV变体进行精确和全面的基因分型。
