| 韩俊娜1, 张继荣1, 旷艺璇1, 王 莹1, 黄爱军1, 2*.西番莲3种病毒多重RT-PCR检测技术的建立与应用[J].植物保护,2025,51(3):289-295. |
| 西番莲3种病毒多重RT-PCR检测技术的建立与应用 |
| Development and application of a multiplex PCR assay for simultaneous detection of three potyviruses in Passiflora edulis |
| 投稿时间：2024-06-27 修订日期：2024-08-20 |
| DOI：10.16688/j.zwbh.2024346 |
| 中文关键词: 西番莲 夜来香花叶病毒 东亚西番莲病毒 西番莲斑驳病毒 多重RT-PCR |
| 英文关键词:Passiflora edulis Telosma mosaic virus East Asian Passiflora virus Passiflora mottle virus multiplex RT-PCR |
| 基金项目:国家自然科学基金(32460646) |
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| 摘要点击次数: 19 |
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| 中文摘要: |
| 西番莲Passiflora edulis是一种广泛分布于我国热带、亚热带地区的高经济作物, 但病毒病害严重制约西番莲的产业发展。尤其马铃薯Y病毒属Potyvirus的夜来香花叶病毒(Telosma mosaic virus, TeMV)、东亚西番莲病毒(East Asian Passiflora virus, EAPV)以及西番莲斑驳病毒(Passiflora mottle virus, PaMoV)是西番莲上发生较为普遍的病毒, 这些病毒单独或者混合侵染均可在西番莲上引起严重危害。本研究针对3种病毒基因组保守区域设计特异引物并筛选, 优化引物浓度、退火温度, 建立了可同时扩增3种病毒的多重RT-PCR检测体系。该检测体系产物片段大小分别是TeMV 685 bp、PaMoV 405 bp和EAPV 248 bp, 扩增产物清晰、特异, 检测TeMV与EAPV的灵敏度分别较普通单重 RT-PCR 灵敏度低约10倍和100倍, PaMoV灵敏度与普通单重RT-PCR灵敏度一致。采用多重RT-PCR和单一RT-PCR对收集到的田间样品分别检测, 结果显示，2种方法检测结果一致, 表明该方法可准确、快速、灵敏地检测复合侵染的3种病毒, 可用于田间发病样品的检测。 |
| 英文摘要: |
| Passiflora edulis is a widely distributed high-value crop in tropical and subtropical regions of China. However, the prevalence of viral diseases represents a significant obstacle to its industrial development. Potyviruses are a common type of virus found on passion fruit, including Telosma mosaic virus (TeMV), East Asian Passiflora virus (EAPV) and Passiflora mottle virus (PaMoV). These viruses, whether alone or in combination, can cause serious damage to passion fruit. In this study, specific primers were designed and screened to target conserved regions of the genomes of the three viruses. The concentrations and annealing temperatures of the primers were optimized, and a multiplex RT-PCR detection system capable of simultaneously amplifying all three viruses was established. The sizes of the amplification products in this detection system were 685 bp for TeMV, 405 bp for PaMoV and 248 bp for EAPV. The amplification products were clear and specific. The sensitivity of TeMV detection was 10 times lower than that of single RT-PCR, while the sensitivity of PaMoV detection was consistent with that of single RT-PCR. The sensitivity of EAPV detection was approximately 100 times lower than that of single RT-PCR. Field samples were tested using both multiplex and single RT-PCR methods, and the results were consistent, indicating that this method can accurately, rapidly, and sensitively detect the three viruses commonly found in mixed infections, which can be recommended for field detection. |
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