Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat

Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores...

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Autores principales: Brusa, Victoria, Galli, Lucía, Linares, Luciano Héctor, Ortega, Emanuel Eneas, Lirón, Juan Pedro, Leotta, Gerardo Aníbal
Formato: Articulo
Lenguaje:Inglés
Publicado: 2015
Materias:
Ciencias Veterinarias
Shiga toxin-producing Escherichia coli
SYBR-PCR
Rt-PCR
stx genes
Ground beef
Acceso en línea:http://sedici.unlp.edu.ar/handle/10915/154157
Aporte de:
SEDICI (UNLP) de Universidad Nacional de La Plata
id I19-R120-10915-154157
record_format dspace
spelling I19-R120-10915-1541572023-06-08T20:09:09Z http://sedici.unlp.edu.ar/handle/10915/154157 issn:0167-7012 Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat Brusa, Victoria Galli, Lucía Linares, Luciano Héctor Ortega, Emanuel Eneas Lirón, Juan Pedro Leotta, Gerardo Aníbal 2015 2023-06-08T18:52:12Z en Ciencias Veterinarias Shiga toxin-producing Escherichia coli SYBR-PCR Rt-PCR stx genes Ground beef Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n = 103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stxpositive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1 × 102 CFU mL−1 limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P b 0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation. Facultad de Ciencias Veterinarias Instituto de Genética Veterinaria Articulo Articulo http://creativecommons.org/licenses/by/4.0/ Creative Commons Attribution 4.0 International (CC BY 4.0) application/pdf 10-17
institution Universidad Nacional de La Plata
institution_str I-19
repository_str R-120
collection SEDICI (UNLP)
language Inglés
topic Ciencias Veterinarias
Shiga toxin-producing Escherichia coli
SYBR-PCR
Rt-PCR
stx genes
Ground beef
spellingShingle Ciencias Veterinarias
Shiga toxin-producing Escherichia coli
SYBR-PCR
Rt-PCR
stx genes
Ground beef
Brusa, Victoria
Galli, Lucía
Linares, Luciano Héctor
Ortega, Emanuel Eneas
Lirón, Juan Pedro
Leotta, Gerardo Aníbal
Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
topic_facet Ciencias Veterinarias
Shiga toxin-producing Escherichia coli
SYBR-PCR
Rt-PCR
stx genes
Ground beef
description Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n = 103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stxpositive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1 × 102 CFU mL−1 limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P b 0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.
format Articulo
Articulo
author Brusa, Victoria
Galli, Lucía
Linares, Luciano Héctor
Ortega, Emanuel Eneas
Lirón, Juan Pedro
Leotta, Gerardo Aníbal
author_facet Brusa, Victoria
Galli, Lucía
Linares, Luciano Héctor
Ortega, Emanuel Eneas
Lirón, Juan Pedro
Leotta, Gerardo Aníbal
author_sort Brusa, Victoria
title Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_short Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_full Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_fullStr Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_full_unstemmed Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_sort development and validation of two sybr green pcr assays and a multiplex real-time pcr for the detection of shiga toxin-producing escherichia coli in meat
publishDate 2015
url http://sedici.unlp.edu.ar/handle/10915/154157
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