PCR detection of enterohemorrhagic escherichia coli o145 in food by targeting genes in the e. coli o145 o-antigen gene cluster and the shiga toxin 1 and shiga toxin 2 genes

Pina M. Fratamico, Chitrita Debroy, Takahisa Miyamoto, Yanhong Liu

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are an important cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Cattle and other animals are potential reservoirs for this pathogen. To develop PCR assays for detection and identification of E. coli O145, the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the O145 O-antigen gene cluster that are specific for this serogroup were selected as targets. Oligonucleotide primers complementary to regions in the E. coli O145 wzx and wzy genes were designed to perform PCR assays with DNA from strains of E. coli O145, non-O145 E. coli serogroups, and other bacterial genera. The assays were highly specific for E. coli O145. A multiplex PCR assay targeting the E. coli O145 wzx and wzy genes and the Shiga toxin 1 (stx 1) and Shiga toxin 2 (stx 2) genes and a real-time multiplex PCR assay targeting the O145 wzy, stx 1, and stx 2 genes were developed for detection of STEC O145. The assays were used for detecting STEC O145 in seeded ground beef, lettuce, and raw milk initially inoculated with ca. 2, 20, or 200 CFU/25g or 25mL after 8 or 20 h of enrichment at 42°C in modified EC broth containing 20 g/L of novobiocin. STEC O145 was detected in all samples inoculated with 2 CFU/25 g or 25. The detection limit of the multiplex PCR assays was ≤7.9times;10 4 CFU/mL, which corresponded to ≤400 CFU/PCR reaction. The PCR assays can be employed to identify enterohemorrhagic E. coli serogroup O145 and to detect low levels of the pathogen in food.

Original languageEnglish
Pages (from-to)605-611
Number of pages7
JournalFoodborne Pathogens and Disease
Volume6
Issue number5
DOIs
Publication statusPublished - Jun 1 2009

Fingerprint

Shiga Toxin 1
Shiga Toxin 2
Shiga-like toxin 1
Shiga-like toxin 2
Enterohemorrhagic Escherichia coli
enterohemorrhagic Escherichia coli
Gene Targeting
gene targeting
Multigene Family
multigene family
Shiga-Toxigenic Escherichia coli
Escherichia coli
antigens
Antigens
Food
Polymerase Chain Reaction
Shiga toxin-producing Escherichia coli
Multiplex Polymerase Chain Reaction
assays
Genes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Animal Science and Zoology

Cite this

PCR detection of enterohemorrhagic escherichia coli o145 in food by targeting genes in the e. coli o145 o-antigen gene cluster and the shiga toxin 1 and shiga toxin 2 genes. / Fratamico, Pina M.; Debroy, Chitrita; Miyamoto, Takahisa; Liu, Yanhong.

In: Foodborne Pathogens and Disease, Vol. 6, No. 5, 01.06.2009, p. 605-611.

Research output: Contribution to journalArticle

@article{f446a6683b51472baaba6080c1d2a0c8,
title = "PCR detection of enterohemorrhagic escherichia coli o145 in food by targeting genes in the e. coli o145 o-antigen gene cluster and the shiga toxin 1 and shiga toxin 2 genes",
abstract = "Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are an important cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Cattle and other animals are potential reservoirs for this pathogen. To develop PCR assays for detection and identification of E. coli O145, the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the O145 O-antigen gene cluster that are specific for this serogroup were selected as targets. Oligonucleotide primers complementary to regions in the E. coli O145 wzx and wzy genes were designed to perform PCR assays with DNA from strains of E. coli O145, non-O145 E. coli serogroups, and other bacterial genera. The assays were highly specific for E. coli O145. A multiplex PCR assay targeting the E. coli O145 wzx and wzy genes and the Shiga toxin 1 (stx 1) and Shiga toxin 2 (stx 2) genes and a real-time multiplex PCR assay targeting the O145 wzy, stx 1, and stx 2 genes were developed for detection of STEC O145. The assays were used for detecting STEC O145 in seeded ground beef, lettuce, and raw milk initially inoculated with ca. 2, 20, or 200 CFU/25g or 25mL after 8 or 20 h of enrichment at 42°C in modified EC broth containing 20 g/L of novobiocin. STEC O145 was detected in all samples inoculated with 2 CFU/25 g or 25. The detection limit of the multiplex PCR assays was ≤7.9times;10 4 CFU/mL, which corresponded to ≤400 CFU/PCR reaction. The PCR assays can be employed to identify enterohemorrhagic E. coli serogroup O145 and to detect low levels of the pathogen in food.",
author = "Fratamico, {Pina M.} and Chitrita Debroy and Takahisa Miyamoto and Yanhong Liu",
year = "2009",
month = "6",
day = "1",
doi = "10.1089/fpd.2008.0254",
language = "English",
volume = "6",
pages = "605--611",
journal = "Foodborne Pathogens and Disease",
issn = "1535-3141",
publisher = "Mary Ann Liebert Inc.",
number = "5",

}

TY - JOUR

T1 - PCR detection of enterohemorrhagic escherichia coli o145 in food by targeting genes in the e. coli o145 o-antigen gene cluster and the shiga toxin 1 and shiga toxin 2 genes

AU - Fratamico, Pina M.

AU - Debroy, Chitrita

AU - Miyamoto, Takahisa

AU - Liu, Yanhong

PY - 2009/6/1

Y1 - 2009/6/1

N2 - Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are an important cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Cattle and other animals are potential reservoirs for this pathogen. To develop PCR assays for detection and identification of E. coli O145, the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the O145 O-antigen gene cluster that are specific for this serogroup were selected as targets. Oligonucleotide primers complementary to regions in the E. coli O145 wzx and wzy genes were designed to perform PCR assays with DNA from strains of E. coli O145, non-O145 E. coli serogroups, and other bacterial genera. The assays were highly specific for E. coli O145. A multiplex PCR assay targeting the E. coli O145 wzx and wzy genes and the Shiga toxin 1 (stx 1) and Shiga toxin 2 (stx 2) genes and a real-time multiplex PCR assay targeting the O145 wzy, stx 1, and stx 2 genes were developed for detection of STEC O145. The assays were used for detecting STEC O145 in seeded ground beef, lettuce, and raw milk initially inoculated with ca. 2, 20, or 200 CFU/25g or 25mL after 8 or 20 h of enrichment at 42°C in modified EC broth containing 20 g/L of novobiocin. STEC O145 was detected in all samples inoculated with 2 CFU/25 g or 25. The detection limit of the multiplex PCR assays was ≤7.9times;10 4 CFU/mL, which corresponded to ≤400 CFU/PCR reaction. The PCR assays can be employed to identify enterohemorrhagic E. coli serogroup O145 and to detect low levels of the pathogen in food.

AB - Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are an important cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Cattle and other animals are potential reservoirs for this pathogen. To develop PCR assays for detection and identification of E. coli O145, the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the O145 O-antigen gene cluster that are specific for this serogroup were selected as targets. Oligonucleotide primers complementary to regions in the E. coli O145 wzx and wzy genes were designed to perform PCR assays with DNA from strains of E. coli O145, non-O145 E. coli serogroups, and other bacterial genera. The assays were highly specific for E. coli O145. A multiplex PCR assay targeting the E. coli O145 wzx and wzy genes and the Shiga toxin 1 (stx 1) and Shiga toxin 2 (stx 2) genes and a real-time multiplex PCR assay targeting the O145 wzy, stx 1, and stx 2 genes were developed for detection of STEC O145. The assays were used for detecting STEC O145 in seeded ground beef, lettuce, and raw milk initially inoculated with ca. 2, 20, or 200 CFU/25g or 25mL after 8 or 20 h of enrichment at 42°C in modified EC broth containing 20 g/L of novobiocin. STEC O145 was detected in all samples inoculated with 2 CFU/25 g or 25. The detection limit of the multiplex PCR assays was ≤7.9times;10 4 CFU/mL, which corresponded to ≤400 CFU/PCR reaction. The PCR assays can be employed to identify enterohemorrhagic E. coli serogroup O145 and to detect low levels of the pathogen in food.

UR - http://www.scopus.com/inward/record.url?scp=67650826055&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67650826055&partnerID=8YFLogxK

U2 - 10.1089/fpd.2008.0254

DO - 10.1089/fpd.2008.0254

M3 - Article

VL - 6

SP - 605

EP - 611

JO - Foodborne Pathogens and Disease

JF - Foodborne Pathogens and Disease

SN - 1535-3141

IS - 5

ER -