A colorimetric, sensitive, rapid, and simple diagnostic kit for the HLB putative causal agent detection

STOLOWICZ, FABIANA; LAROCCA, LUCIANA; WERBAJH, SANTIAGO; PARMA, YANIL; CARRILLO, CAROLINA; OGAS, LORENA; AGOSTINI, JUAN PEDRO; REDES, JONATHAN; WELIN, BJORN; CASTAGNARO, ATILIO; VOJNOV, ADRIAN

Frontiers in Agronomy

Frontiers

Lugar: Lausanne; Año: 2022 vol. 4 p. 1 - 11

Huanglongbing (HLB) is one of the most devastating diseases in citrus worldwide.The Gram-negative bacterial plant pathogen “Candidatus Liberibacter spp.” isphloem-limited and vectored by citrus psyllids. The species “CandidatusLiberibacter asiaticus” (C.Las) has been detected in Argentina, and its vector hasbeen found in at least nine provinces. Early detection of C.Las is critical for asuccessful management of HLB disease. Currently, HLB molecular diagnosis iscarried out by PCR, nested PCR, real-time PCR, or another combination of thesetechniques, which require purification of genomic DNA, sophisticated equipment,and highly trained personnel. We have developed a prototype of a sensitivecolorimetric kit to detect C.Las based on the specific DNA isothermalamplification of this microorganism. The reaction buffer containshydroxynaphthol blue (HNB), an indicator dye that turns from violet to blue/lightblue when the DNA amplification reaction is positive. Similar sensitivity to visualize apositive reaction was observed between HNB loop-mediated isothermalamplification and agarose gel electrophoresis analysis. The detection of C.Lasinfected plants was up to 8 ng of total infected plant genomic DNA, similar toquantitative PCR. A blind validation test of the prototype kit was performed withpurified DNA extracted from healthy orC.Las-infectedmidrib plants. Our kit showed100% concordance with the results of a gold-standard quantitative PCR techniqueapplied by the Laboratorio de Biologıa Molecular de EEA Montecarlo. The analysis of ́samples, without DNA purification to detect C.Las, showed a similar sensitivity to theanalysis of the same samples in which C.Las DNA was previously purified.