title: A cultivation-independent PCR-RFLP assay targeting oprF gene for detection and identification of Pseudomonas spp. in samples from fibrocystic pediatric patients.
abstract: Species-specific genetic markers are crucial to develop faithful and sensitive molecular methods for the detection and identification of Pseudomonas aeruginosa (Pa). We have previously set up a PCR-RFLP protocol targeting oprF, the gene encoding the genus-specific outer membrane porin F, whose strong conservation and marked sequence diversity allowed detection and differentiation of environmental isolates (Agaras et al., 2012). Here, we evaluated the ability of the PCR-RFLP assay to genotype clinical isolates previously identified as Pa by conventional microbiological methods within a collection of 62 presumptive Pa isolates from different pediatric clinical samples and different sections of the Hospital de Niños "Sor María Ludovica" from La Plata, Argentina. All isolates, but one, gave an oprF amplicon consistent with that from reference Pa strains. The sequence of the smaller-sized amplicon revealed that the isolate was in fact a mendocina Pseudomonas strain. The oprF RFLP pattern generated with TaqI or HaeIII nucleases matched those of reference Pa strains for 59 isolates (96%). The other two Pa isolates (4%) revealed a different RFLP pattern based on HaeIII digestion, although oprF sequencing confirmed that Pa identification was correct. We next tested the effectiveness of the PCR-RFLP to detect pseudomonads on clinical samples of pediatric fibrocystic patients directly without sample cultivation. The expected amplicon and its cognate RFLP profile were obtained for all samples in which Pa was previously detected by cultivation-dependent methods. Altogether, these results provide the basis for the application of the oprF PCR-RFLP protocol to directly detect and identify Pa and other non-Pa pseudomonads in fibrocystic clinical samples.
authors.affiliation: Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal B1876BXD, Buenos Aires, Argentina.
authors.forename: Antonio
authors.initials: A
authors.lastname: Lagares
authors.affiliation: Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal B1876BXD, Buenos Aires, Argentina.
authors.forename: Betina
authors.initials: B
authors.lastname: Agaras
authors.affiliation: Sala de Microbiología HIAEP "Sor María Ludovica", Calle 14N° 1631, La Plata B1904CSI, Buenos Aires, Argentina.
authors.forename: Marisa P
authors.initials: MP
authors.lastname: Bettiol
authors.affiliation: Sala de Microbiología HIAEP "Sor María Ludovica", Calle 14N° 1631, La Plata B1904CSI, Buenos Aires, Argentina.
authors.forename: Blanca M
authors.initials: BM
authors.lastname: Gatti
authors.affiliation: Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal B1876BXD, Buenos Aires, Argentina. Electronic address: cvalver@unq.edu.ar.
authors.forename: Claudio
authors.initials: C
authors.lastname: Valverde
mesh_terms: D001118:Argentina; D001426:Bacterial Proteins / Q000235:genetics*; D003550:Cystic Fibrosis / Q000150:complications*; D004269:DNA, Bacterial / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011552:Pseudomonas Infections / Q000175:diagnosis* / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C547605:OprF protein, Pseudomonas aeruginosa; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; C027046:TCGA-specific type II deoxyribonucleases
keywords: Cystic fibrosis; PCR-RFLP; Pseudomonas aeruginosa; oprF