Three duplex real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) assays based on TaqMan chemistry, were developed for the simultaneous detection and specific quantification of apple chlorotic leafspot virus (ACLSV), plum pox virus (PPV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), peach latent mosaic viroid (PLMVd) and the European stone fruit yellows (ESFY) phytoplasma, which are considered among the most important pathogens affecting stone fruit trees. The quantitative RT-PCR (RT-qPCR) assays were optimized using RNA transcripts (linearized plasmid was used for the assay optimization of the ESFY phytoplasma) of known concentrations. No differences in sensitivity were recorded between the duplex and singleplex RT-qPCR assays. The amplification efficiency of the duplex assays reached 91.1–95.8%, while the linear range of quantification was from 20 to 2 × 10⁷ RNA/linearized plasmid transcripts for PLMVd and ESFY phytoplasma, 40 to 4 × 10⁷ RNA transcripts for ACLSV, PPV and PDV, and 10² to 10⁸ RNA transcripts for PNRSV, respectively. The duplex RT-qPCR assays, which were validated using both characterized isolates from all pathogens and field samples from Prunus species in Northern Greece, exhibited a broad detection range. Overall, the developed methods comprise useful tools that could be applied for the simultaneous and reliable detection of graft-transmissible pathogens in certification programs of Prunus spp.

开发了基于TaqMan化学的三种双链体实时逆转录聚合酶链反应（实时RT-PCR）分析方法，用于同时检测和定量苹果绿化叶斑病毒（ACLSV），李子痘病毒（PPV），李属坏死环斑病毒（PNRSV），西梅矮毒（PDV），桃潜在花叶病毒（PLMVd）和欧洲核果黄（ESFY）植物质体，被认为是影响核果树的最重要病原体。使用已知浓度的RNA转录本（线性化质粒用于ESFY植物质体的测定优化）优化了定量RT-PCR（RT-qPCR）测定。在双工和单重RT-qPCR分析之间未记录灵敏度差异。双重分析的扩增效率达到91.1–95.8％，而线性定量范围为PLMVd和ESFY植物质体的线性范围为20至2×10 ⁷ RNA /线性化质粒转录物，ACLSV，PPV的线性范围为40至4×10 ⁷ RNA转录物和PDV，以及10PNRSV的²至10 ^8个RNA转录本。使用来自所有病原体的特征分离株和来自希腊北部李属物种的野外样品进行验证的双工RT-qPCR分析显示了广泛的检测范围。总体而言，已开发的方法包括有用的工具，这些工具可用于在李属鉴定程序中同时可靠地检测移植物可传播的病原体。