title: Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces.
abstract: A conventional PCR targeted directly to the detection of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stools of symptomatic patients may require the introduction of internal controls to detect false negative results. In the present study, we designed a competitive internal amplification control (IAC) to be included in a well-known PCR protocol used to amplify the stx1and stx2 genes from STEC isolates. The IAC was introduced in the PCR reaction and amplified when E. coli O157:H7 cultures and contaminated pediatric feces were assayed. When STEC concentration was 10(3) CFU ml(-1) in pure culture and 10(4) CFU g(-1) in contaminated stools, the IAC at concentration of 0.143 pg μl(-1) in the PCR reaction mixture was co-amplified with the stx2 sequence, producing bands of 279 and 349 bp, respectively. These STEC values were considered the detection limits of the duplex PCR. The specific detection of STEC by duplex PCR including IAC might be achieved directly on pediatric feces when the pathogen load reaches concentrations of at least 10(4) CFU g(-1).
authors.affiliation: Microbiología General, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina. Electronic address: gabo.3333@gmail.com.
authors.forename: Ángel Gabriel
authors.initials: ÁG
authors.lastname: Salinas-Ibáñez
authors.affiliation: Microbiología General, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina; Instituto Multidisciplinario de Investigaciones Biológicas de San Luis-Consejo de Investigaciones Científicas y Tecnológicas (IMIBIO-CONICET), Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Lucero-Estrada
authors.affiliation: Biología Molecular, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina; Instituto de Biología Agrícola de Mendoza (IBAM), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Cuyo (FCA-UNCuyo), Almirante Brown 500, M5528AHB Chacras de Coria, Mendoza, Argentina.
authors.forename: Constanza
authors.initials: C
authors.lastname: Chialva
authors.affiliation: Biología Molecular, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina.
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: Zárate
authors.affiliation: Biología Molecular, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina; Instituto Multidisciplinario de Investigaciones Biológicas de San Luis-Consejo de Investigaciones Científicas y Tecnológicas (IMIBIO-CONICET), Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina.
authors.forename: Maximiliano
authors.initials: M
authors.lastname: Juri-Ayub
authors.affiliation: Microbiología General, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina.
authors.forename: María Esther
authors.initials: ME
authors.lastname: Escudero
mesh_terms: D002675:Child, Preschool; D003967:Diarrhea / Q000382:microbiology; D004927:Escherichia coli Infections / Q000175:diagnosis; D005188:False Negative Reactions; D005243:Feces / Q000382:microbiology*; D006801:Humans; D007223:Infant; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D022622:Shiga Toxin 1 / Q000235:genetics*; D022641:Shiga Toxin 2 / Q000235:genetics*; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*
chemical_list: D022622:Shiga Toxin 1; D022641:Shiga Toxin 2
keywords: EHEC; Escherichia coli; Internal amplification control; PCR; Pediatric feces; STEC