李痘病毒实时荧光定量RT-PCR检测方法的建立及应用 Click here to download the full text Citation：邢飞,夏思丹,金畑宏,邹国臣,淳长品,李世访.李痘病毒实时荧光定量RT-PCR检测方法的建立及应用.Journal of Plant Protection,2026,53(4):989-997 Hits: Download times: Author Name Affiliation E-mail Xing Fei  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Xia Sidan  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Jin Tianhong  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Zou Guochen  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Chun Changpin  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Li Shifang  Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China  sfli@ippcaas.cn  中文摘要:为高效检测检疫性植物病毒李痘病毒（plum pox virus，PPV），根据其外壳蛋白（coat protein，CP）基因的保守序列设计特异性引物与荧光探针，建立基于TaqMan荧光探针的PPV实时荧光定量反转录-PCR（reverse transcription PCR，RT-PCR）检测技术，并分析其重复性、灵敏性、特异性和实际应用效果。结果表明：当引物浓度为0.6 μmol/L、TaqMan探针浓度为0.2 μmol/L时，所建实时荧光定量RT-PCR技术体系的扩增信号较好；重复性试验的变异系数较小，为0.61%~1.59%，重复性较好；该方法对PPV的检测灵敏度是常规RT-PCR方法的100倍，且与目前已知侵染李属Prunus植物的主要病毒没有交叉反应，说明特异性良好。利用所建方法检测了30份梅P.mume和80份脆李P.salicina叶片样品中PPV侵染情况，其中在28份表现典型李痘病症状的梅样品中均检出PPV，在无明显李痘病症状的梅和脆李样品中均没有检出PPV。表明本研究建立的实时荧光定量RT-PCR技术体系适用于PPV的快速、高效检测，可用于田间该病毒的有效检测。 中文关键词:李痘病毒  实时荧光定量RT-PCR  TaqMan探针  梅  脆李   Establishment and application of reverse transcription-real-time fluorescence quantitative PCR detection system for plum pox virus Author Name Affiliation E-mail Xing Fei  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Xia Sidan  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Jin Tianhong  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Zou Guochen  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Chun Changpin  National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China    Li Shifang  Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China  sfli@ippcaas.cn  Abstract:To achieve efficient detection of plum pox virus (PPV)，a quarantine plant virus, we developed a reverse transcription-real-time fluorescence quantitative PCR (RT-qPCR) assay based on the TaqMan probe. Specific primers and the fluorescent probe were designed based on the conserved region of the coat protein (CP) gene of PPV. The repeatability, sensitivity, specificity, and practical application performance of the assay were evaluated. The results showed that optimal amplification signal was obtained when the primer and the TaqMan probe concentrations were 0.6 μmol/L and 0.2 μmol/L, respectively. Repeatability analysis indicated that the coefficients of variation ranged from 0.61% to 1.59%, demonstrating low variability and high reproducibility. The sensitivity of this method for PPV detection was 100-fold higher than that of conventional RT-PCR, and no cross-reactivity was observed with other major viruses known to infect the Prunus plants, confirming its high specificity. The established method was subsequently applied to detect PPV infection in 30 Japanese apricot (Prunus mume) leaf samples and 80 Chinese plum (Prunus salicina) leaf samples. PPV was detected in all 28 Japanese apricot leaf samples with typical plum pox symptoms, but none of the asymptomatic Japanese apricot samples and all Chinese plum leaf samples tested positive for PPV. These results suggest that the developed RT-qPCR system in this study is suitable for the rapid and efficient detection of PPV and could be employed for field surveillance of this virus. keywords:plum pox virus  real-time fluorescence quantitative RT-PCR  TaqMan probe  Prunus mume  Prunus salicina View Full Text  View/Add Comment  Download reader

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