Novel set of real-time PCR primers for simultaneous detection of Liberibacter

INGRID GEORGINA ORCE,; LORENA NOELIA SENDÍN; MARÍA ROSA MARANO; ADRIÁN ALBERTO VOJNOV; ATILIO PEDRO CASTAGNARO, ; MARÍA PAULA FILIPPONE

SCIENTIA AGRICOLA

UNIV SAO PAOLO

Lugar: Piracicaba; Año: 2015

0103-9016

Huanglongbing (HLB), a devastating citrus disease caused by the bacterium ?CandidatusLiberibacter spp.?, is now responsible for significant economic losses worldwide. Yet, noeffective disease control has been found, and the non-cultivability of the bacterium has severelyhampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essentialfor cell survival, and is used for bacterial identification or assignment of close relationshipsat the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNAgenes are widely used in the detection of ?Ca. Liberibacter spp.? in multiplex reactions. We havedeveloped 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 speciesassociated with HLB, and can differentiate Ca. Liberibacter asiaticus or africanus from americanusby their characteristic melting curves. The assay is very sensitive, and it was possibleto amplify expected DNA fragments with an efficiency of 98 % using the Syber Green systemand a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simpleand efficient detection methodology could also be important in the detection of all species ofHLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step inthe development of preventive strategies aimed at avoiding the dissemination of this devastatingdisease in HLB-free areas.