Bertacca Sofia, Caruso Andrea Giovanni, Trippa Daniela, Marchese Annalisa, Giovino Antonio, Matic Slavica, Noris Emanuela, Ambrosio Maria Isabel Font San, Alfaro Ana, Panno Stefano, Davino Salvatore
Department of Agricultural, Food and Forest Sciences (SAAF), University of Palermo, Viale delle Scienze, 90128 Palermo, Italy.
Research Centre for Plant Protection and Certification (CREA), Strada Statale, 113, 90011 Bagheria, Italy.
Plants (Basel). 2022 Feb 28;11(5):660. doi: 10.3390/plants11050660.
A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material.
开发了一种实时环介导等温扩增（LAMP）检测方法，用于简单、快速且高效地检测油橄榄双生病毒（OEGV），该病毒最近在全球不同橄榄种植区被报道。通过终点PCR对OEGV进行初步筛查以确定其在西西里岛的存在情况。设计了一组六条实时LAMP引物，靶向OEGV编码外壳蛋白（AV1）基因区域的一段209个核苷酸的序列，用于特异性检测OEGV。测定了该诊断检测方法的特异性、灵敏度和准确性。LAMP检测方法与其他双生病毒无交叉反应，并且检测OEGV的灵敏度比传统终点PCR高10倍。为提高LAMP检测方法用于现场诊断的潜力，建立了一种简化的样品制备程序，并用于监测西西里岛不同橄榄品种中OEGV的传播情况。作为此次调查的结果，我们观察到在分析的70个品种中有30个对OEGV呈阳性，表明OEGV发病率相对较高。本研究中开发的实时LAMP检测方法适用于设施和资源有限的植物病理学实验室，也适用于现场直接检测OEGV，是一种快速筛查橄榄植物材料的可靠方法。
