Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10957676 | Molecular and Cellular Probes | 2015 | 29 Pages |
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 103 CFU mlâ1 in pure culture and 104 CFU gâ1 in contaminated stools, the IAC at concentration of 0.143 pg μlâ1 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 gâ1.
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Authors
Ángel Gabriel Salinas-Ibáñez, Cecilia Lucero-Estrada, Constanza Chialva, Juan Manuel Zárate, Maximiliano Juri-Ayub, MarÃa Esther Escudero,