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.

Accuracy of reverse‐transcription polymerase chain reaction and loop‐mediated isothermal amplification in diagnosing severe fever with thrombocytopenia syndrome: A systematic review and meta‐analysis

“…In our study, the pooled sensitivity, specificity, PLR, NLR, and AUC of RT‐LAMP were 0.95, 0.99, 111.18, 0.05, and 0.99, respectively, which also indicates a high accuracy. RT‐LAMP can qualitatively determine SFTS infection by identifying discoloration and precipitation, 34 making it suitable for developing regions, primary care facilities, small clinical laboratories, and other applications in the diagnosis of various viral diseases 40–42 . Even if the conditions are limited, primary clinical decisions can still be implemented.…”

“…RT‐LAMP can qualitatively determine SFTS infection by identifying discoloration and precipitation, 34 making it suitable for developing regions, primary care facilities, small clinical laboratories, and other applications in the diagnosis of various viral diseases. 40 , 41 , 42 Even if the conditions are limited, primary clinical decisions can still be implemented. However, for use in scientific research, more sensitive multiplex detection experiments still need be developed for the more rapid detection of multiple viral diseases, as demonstrated in the study by Jang et al, 16 in which the simultaneous detection of SFTS and scrub typhus was attempted.…”

Fast and Sensitive Detection of Soil-Borne Cereal Mosaic Virus in Leaf Crude Extract of Durum Wheat

“…Furthermore, its sensitivity is higher or similar to that of other molecular techniques, allowing the detection of small quantities of the target viral genome (picograms to femtograms), depending on the host/virus combination [ 26 , 27 , 28 , 29 ]. In addition, the high resistance of the LAMP Bst DNA polymerase enzyme to reaction inhibitors [ 30 , 31 ] makes it possible to skip the RNA or DNA extraction step and use directly the crude plant extracts in which tissues are mechanically disrupted and homogenised in a buffer [ 32 , 33 ].…”

In-Field LAMP Detection of Flavescence Dorée Phytoplasma in Crude Extracts of the Scaphoideus titanus Vector

“…Among these techniques, LAMP [28] is widely used in plant pathology thanks to the speed of performance and the rapidity in obtaining results [29]. Furthermore, its sensitivity is higher or comparable to other molecular techniques, allowing the detection of the target DNA in quantities ranging from picograms to femtograms, depending on the protocol [30,31].…”