VALIDATION OF A FAST AND SIMPLE DIAGNOSTIC KIT FOR HLB CAUSAL AGENT DETECTION

STOLOWICZ FABIANA; LAROCCA LUCIANA; WERBAJH SANTIAGO; PARMA YANIL; AGOSTINI JP; REDES J; CARRILLO CAROLINA; VOJNOV ADRIAN

Mar Del Plata

Congreso; XXI Reunión Anual de la sociedad Argentina de Biología (SAB) 2019; 2019

SAB

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 performed by PCR, Nested PCR, real time PCR or some combination of them, requiring purified genomic DNA, sophisticated equipment and qualified human resources.The aim of this study was to evaluate the performance of a sensible, fast and simple diagnosis test based on specific DNA isothermal amplification of Ca. L asiaticus by comparison with PCR and qPCR, considered the Gold standard methods to HLB diagnosis. We applied the test in a group of samples whose true disease status was defined by the mentioned gold standard techniques. Analyzing the results by a 2 x 2 contingency table, we determined Sensitivity and Specificity of the test, and the positive and negative predictive values (PPV and NPV). In a first test, 30 DNA samples were analyzed and compared with qPCR technique with a concordance in 28 samples (PPV and NPV of 100% and 88,88%, respectively). Adjusting test parameters of reading out, 23 new samples consisting on midribs and purified genomic DNA from uninfected or Ca. L. asiaticus infected plant lines were analyzed in a blind assay comparing with PCR/Nested PCR applied by the Molecular Laboratory of the EEA Montecarlo (INTA), obtaining 100% of concordance (PPV and NPV, both 100%).The results obtained in the present study demonstrated a high quality of our diagnostic test, with low cost, making it a valid and useful tool to support the diagnosis of HLB disease.