Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus

Abstract

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.