Validation of a fast and simple diagnostic kit for HLB causal agent detection

STOLOWICZ FG; LAROCCA L; WERBAJH S; PARMA Y; AGOSTINI JP; REDES J; CARRILLO C; VOJNOV AA

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias; 2019

SAP, SAB, SAFE, SAIC

The Huanglongbing (HLB), is the most devastating disease in citrus worldwide, due to the damage it causes, the difficulty of diagnosis and the speed of its expansion. The causal pathogen, Candidatus Liberibacter spp (Ca. L), is transmitted by the insect Diaphorina citri. The species Ca. L asiaticus has been detected in Argentina, and the vector distributed at least in nine provinces. Since 2010, the Argentinian National Service for Health and Agro-Food Quality (SENASA), has implemented a National HLB Prevention Program to safeguard productivity in this important sector.Nowadays, HLB diagnosis is made by PCR, Nested PCR, real time PCR or some combination of them, requiring purified genomic DNA, sophisticated equipment and qualified human resources.Our aim is to developed a sensible, fast and simple diagnosis technique based on specific DNA isothermal amplification, usable in any field condition. In first steps, we developed a simple test that only required any kind of a heater (stable temperature at 64°C), and tested samples of purified plant genomic DNA, uninfected or infected with HLB, finding a result concordance in 28 of 30 samples analyzed by a gold standard qPCR Taqman (the EEAOC method). Based on those results, we adjusted some variables of the method and obtained a prototype kit usable with unpurified DNA samples and with a simple eye-naked read out. To evaluate the prototype performance, 23 samples consisting on midribs or purified genomic DNA from uninfected or Ca. L. asiaticus infected plant lines were analyzed in a blind assay. The gold standard technique was the PCR/Nested PCR applied by the Laboratorio de Biología Molecular de EEA Montecarlo, obtaining 100% of concordance. As the simplified prototype kit shows adequate sensitivity and reproducibility, it encourages us to advance with a bigger studio under field conditions to validate the kit in view to transferring it to market.