title: A PCR-Based Diagnostic System for Differentiating Two Weevil Species (Coleoptera: Curculionidae) of Economic Importance to the Chilean Citrus Industry.
abstract: A PCR-based method was developed to identify Naupactus cervinus (Boheman) and Naupactus xanthographus (Germar), two curculionids affecting the citrus industry in Chile. The quarantine status of these two species depends on the country to which fruits are exported. This identification method was developed because it is not possible to discriminate between these two species at the egg stage. The method is based on the species-specific amplification of sequences of internal transcribed spacers, for which we cloned and sequenced these genome fragments from each species. We designed an identification system based on two duplex-PCR reactions. Each one contains the species-specific primer set and a second generic primer set that amplify a short 18S region common to coleopterans, to avoid false negatives. The marker system is able to differentiate each Naupactus species at any life stage, and with a diagnostic sensitivity to 0.045 ng of genomic DNA. This PCR kit was validated by samples collected from different citrus production areas throughout Chile and showed 100% accuracy in differentiating the two Naupactus species.
authors.affiliation: Biotechnology Lab, La Platina Experimental Center, Instituto de Investigaciones Agropecuarias (INIA), Santa Rosa 11,610, Santiago, 8820000, Chile. Doctoral Program in Agricultural and Veterinary Sciences, Universidad de Chile, Santa Rosa 11,315, Santiago, 8820000, Chile.
authors.forename: C
authors.initials: C
authors.lastname: Aguirre
authors.affiliation: La Cruz Experimental Center, Instituto de Investigaciones Agropecuarias (INIA), Chorrillos 86, La Cruz, Quillota, 2260000, Chile.
authors.forename: N
authors.initials: N
authors.lastname: Olivares
authors.affiliation: La Cruz Experimental Center, Instituto de Investigaciones Agropecuarias (INIA), Chorrillos 86, La Cruz, Quillota, 2260000, Chile.
authors.forename: P
authors.initials: P
authors.lastname: Luppichini
authors.affiliation: Biotechnology Lab, La Platina Experimental Center, Instituto de Investigaciones Agropecuarias (INIA), Santa Rosa 11,610, Santiago, 8820000, Chile. phinrichsen@inia.cl.
authors.forename: P
authors.initials: P
authors.lastname: Hinrichsen
mesh_terms: D000818:Animals; D002677:Chile; D002957:Citrus; D021901:DNA, Intergenic; D016133:Polymerase Chain Reaction; D048889:Weevils / Q000145:classification* / Q000235:genetics
chemical_list: D021901:DNA, Intergenic
keywords: Chile; Naupactus cervinus; Naupactus xanthographus; citrus industry; internal transcribed spacer of nuclear ribosomal DNA