Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces.
Mol Cell Probes
; 29(6): 351-357, 2015 Dec.
Article
en En
| MEDLINE
| ID: mdl-26416409
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 O157H7 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).
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Toxina Shiga I
/
Toxina Shiga II
/
Escherichia coli Shiga-Toxigénica
/
Heces
/
Reacción en Cadena de la Polimerasa Multiplex
Tipo de estudio:
Diagnostic_studies
Límite:
Child, preschool
/
Humans
/
Infant
Idioma:
En
Revista:
Mol Cell Probes
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Año:
2015
Tipo del documento:
Article