info:eu-repo/semantics/article
Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces
Fecha
2015-12Registro en:
Salinas Ibáñez, Ángel Gabriel; Lucero Estrada, Cecilia Stella Marys; Chialva, Constanza Soledad; Zárate, Juan Manuel; Juri Ayub, Maximiliano; et al.; Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces; Academic Press Ltd - Elsevier Science Ltd; Molecular And Cellular Probes; 29; 6; 12-2015; 351-357
0890-8508
CONICET Digital
CONICET
Autor
Salinas Ibáñez, Ángel Gabriel
Lucero Estrada, Cecilia Stella Marys
Chialva, Constanza Soledad
Zárate, Juan Manuel
Juri Ayub, Maximiliano
Escudero, María Esther
Resumen
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 103 CFU ml-1 in pure culture and 104 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 104 CFU g-1.