LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP): A SIMPLE METHOD FOR EARLY DETECTION OF THE Xanthomona arboricola SPECIES

PAZ E; FERNÁNDEZ BALDO M; TESÁN I; RINALDI TOSI ME; GARRO N

Mendoza

Congreso; XXXVI REUNIÓN CIENTIFICA ANUAL DE LA SOCIEDAD DE BIOLOGÍA DE CUYO; 2018

Sociedad de Biología de Cuyo

Xanthomona arboricola (Xa) species are the major cause for premature drop fruit in walnuts, hazelnutus, almonds and other fruits dried, and responsible to of great economic losses throughout the world. On the over hand, Loop-mediated isothermal amplification (LAMP) is a simple, specific, economic and rapid nucleic acid amplification method compared to the conventional PCR assay. This study aims to develop methods that allow early diagnosis, anticipate infection by microorganisms and act quickly on plants health and control strategies. For this purpose, different target genes such as XopA, XopAH and XopG were studied together with various primers for both isothermal amplification and conventional PCR using Bioedit and PrimeExplore V4 software respectively. Thus, samples of bacteria were isolated from infected tissues, from which the genomic material was extracted using column purification techniques and direct isolation of colonies, and kept at -20°C until its used. For LAMP-PCR asssays external primers (F3-B3), inside primers (FIP-BIP) and Bst 2.0 warm start polymerase, and for conventional PCR assays, commun primers and Taq polymerase were used. Amplifications were detected in 1.8% agarose gel, which showed two characteristics fragments that were compared with a ladder. To compare results, we used conventional PCR primers (LF and LB), the latter was also performed to verify the functioning of the primers to be used in LAMP-PCR. Amplifications for LAMP-PCR were detected by simple visualization with turbidimetric method by magnesium pyrophosphate precipitate formation and fluorometric method by addition of ethidium bromide in both reaction tubes. The results of LAMP-PCR assay obtained during the analysis of nut samples from different regions of Rio Negro valley showed that XopA and XopAH genes were positive after 25 minutes reaction and XopG gene negative results. Moreover, the same genes showed positive results in conventional PCR assay with similar specificity. We concluded that the LAMP-PCR cualitative assay can be used to detect different genes that recognize microorganisms responsible for premature drop and thus make early detection before that occur physic manifestation of the symptomatology. It also does not require a thermal cycler and is an excellent tool for the development of diagnostic kit.