Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes

Pina M. Fratamico, Chitrita Debroy

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx 1, stx 2, and wzy O157 genes in combination with probes and primers targeting either the fliC h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx 1, stx 2, wzy O157, and eae genes; however, using the assay targeting the stx 1, stx 2, wzy O157, and fliC h7 gene combination, a positive result was always obtained for the fliC h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx 1, stx 2, eae, and wzy O157 or the fliC h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.

Original languageEnglish (US)
Pages (from-to)330-337
Number of pages8
JournalFood Analytical Methods
Volume3
Issue number4
DOIs
StatePublished - Apr 22 2010

Fingerprint

Escherichia coli O157
Multiplex Polymerase Chain Reaction
Escherichia coli
Real-Time Polymerase Chain Reaction
Assays
Genes
food
Food
Beef
assays
ground beef
genes
apple cider
Lettuce
Stem Cells
Malus
raw milk
lettuce
Milk
food enrichment

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Food Science
  • Applied Microbiology and Biotechnology
  • Safety, Risk, Reliability and Quality
  • Safety Research

Cite this

@article{f7a0479840c04582b584aa8c9015c100,
title = "Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes",
abstract = "Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx 1, stx 2, and wzy O157 genes in combination with probes and primers targeting either the fliC h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx 1, stx 2, wzy O157, and eae genes; however, using the assay targeting the stx 1, stx 2, wzy O157, and fliC h7 gene combination, a positive result was always obtained for the fliC h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx 1, stx 2, eae, and wzy O157 or the fliC h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.",
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Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes. / Fratamico, Pina M.; Debroy, Chitrita.

In: Food Analytical Methods, Vol. 3, No. 4, 22.04.2010, p. 330-337.

Research output: Contribution to journalArticle

TY - JOUR

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N2 - Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx 1, stx 2, and wzy O157 genes in combination with probes and primers targeting either the fliC h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx 1, stx 2, wzy O157, and eae genes; however, using the assay targeting the stx 1, stx 2, wzy O157, and fliC h7 gene combination, a positive result was always obtained for the fliC h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx 1, stx 2, eae, and wzy O157 or the fliC h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.

AB - Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx 1, stx 2, and wzy O157 genes in combination with probes and primers targeting either the fliC h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx 1, stx 2, wzy O157, and eae genes; however, using the assay targeting the stx 1, stx 2, wzy O157, and fliC h7 gene combination, a positive result was always obtained for the fliC h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx 1, stx 2, eae, and wzy O157 or the fliC h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.

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