Loop mediated isothermal amplification: an innovative gene amplification technique for plant diseases


1Agricultural University of Tirana, Faculty of Agriculture and Environment/ Rruga “Paisi Vodica, Kodër-Kamëz, Tiranë, Albania

2C.I.H.E.A.M. – Istituto Agronomico Mediterraneo di Bari/ Via Ceglie 9, 70010 Valenzano (BA), Italy. 

3University of Zagreb Faculty of Agriculture, Svetošimunska 25, 10000 Zagreb, Croatia

*Corresponding author  e-mail: magdacara@ubt.edu.al        

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Nowadays, molecular diagnostic methods of plant pathogens evolved in a fast way, the use of rapid and easy to use detection method is fundamental to prevent pathogens cross border and spread to enhance food quality and security. Real – Time Loop-mediated isothermal amplification (RT-LAMP) is a novel molecular detection method that specifically detects genomic DNA by using a set of six oligonucleotide primers specific to different regions of a target gene and Bacillus stearothermophilus (Bst) DNA polymerase protein. This method has been recently modified to be use as a RT-LAMP and then widely applied in many fields for on-site detection and ability to be used in cross border control of plant health, such as quarantine disease diagnosis. The application of rapid and simple DNA extraction method (10 min at 65°C) shortened the detection assay to less than one hour. During the period 2015–2017 are tested in Albania more than 100 samples for: Xylella fastidiosa in olive trees, Flavescence doree in vineyards. Olive samples were taken in Vlora, Durrës, Saranda, etc. All the samples have been negative. Samples for the Flavescence doree have been taken in Lezhë, Vlorë, Durrës in Albania and Istria in Croatia. The samples from Albania resulted negative while the samples from Croatia (17 samples) were 53% positive. Every procedure has been confirmed with positive and negative controls to use diagnostic kit denominated Xylella Screen Glow & FD from Enbiotech s.r.l., Italy. RT-LAMP method is more sensitive than convectional and RT PCR, advantage of LAMP is the isothermal reaction condition, hereby LAMP is affordable because of no need to have expensive thermal cycler. Although recommended reagent storage temperature is -20oC, reagents can be stored at environment temperature. Hereby there is no need to have cold chain for reagent distribution. The results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive and specific and has potential application in plant pathology surveillance. This new method is widely used nowadays by many laboratories and is recognized by EPPO as a standard method for some pathogens. Test performance study is being developed today in the circles of European scientists.

Keywords: Loop mediated isothermal amplification; Bst DNA polymerase, plant diseases.

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