Novel set of real-time PCR primers for simultaneous detection of Liberibacter species associated with citrus Huanglongbing

ORCE I.G.; SENDÍN L.N.; MARANO M.R.; VOJNOV A.A.; CASTAGNARO A.P. ; FILIPPONE M.P.

SCIENTIA AGRICOLA

UNIV SAO PAOLO

Año: 2015 vol. 72 p. 252 - 259

0103-9016

Huanglongbing (HLB), a devastating citrus disease caused by the bacterium ?Candi- datus Liberibacter spp.?, is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essen- tial for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of ?Ca. Liberibacter spp.? in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiate Ca. Liberibacter asiaticus or africanus from ameri- canus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.