Differentiation of stx1A gene for detection of Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 in food samples using high resolution melting curve analysis.

Babak Pakbin,Afshin Akhondzadeh Basti,Abdollah Karimi,Leila Azimi,Ali Khanjari

doi:10.1002/fsn3.1649

Babak Pakbin, Afshin Akhondzadeh Basti + Show 3 more

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https://doi.org/10.1002/fsn3.1649

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Abstract

Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 as the Shiga toxin‐producing bacteria cause some acute gastrointestinal and extraintestinal diseases such as hemorrhagic uremic syndrome and bloody diarrhea in human. Stx genes are the key virulence factors in these pathogens. The aim of this study was to develop HRMA assay to differentiate stx1A gene for detection of E. coli serotype O157: H7 and Sh. dysenteriae type 1 and determine the prevalence of these pathogens in food samples using this method. PCR‐HRMA assay and gold standard methods have been carried out for identification of pathogens among 135 different food samples. We found HRMA method a sensitive and specific assay (100 and 100%, respectively) for differentiation of stx1A gene, consequently, detection of these pathogens in food samples. Also, the highest prevalence of E. coli serotype O157: H7 and Sh. dysenteriae type 1 harboring stx1A gene was observed in raw milk and vegetable salad samples, respectively. HRMA as a rapid, inexpensive, sensitive and specific method is suggested to be used for differentiation of stx1A gene to detect E. coli serotype O157: H7 and Sh. dysenteriae type 1 as the key pathogens for safety evaluation of food samples.

Highlights

The goal of this study was to develop high resolution melting curve analysis (HRMA) polymerase chain reaction (PCR) assay for differentiation and detection of stx1A gene to identify E. coli O157: H7 and Sh. dysenteriae type 1 isolated from food samples and determination of prevalence of these pathogens by this method
Specific primer pairs for identification of E. coli serotype O157: H7 and Sh. dysenteriae type 1 were designed based on detection of stx1A gene-encoded Shiga toxin released by these pathogens using IDT PrimerQuest online tool to design specific primers for qPCR intercalating dye reaction (IDT, USA)
3.1 | Identification of stx1A using HRMA method and assessment of the assay stx1A gene for detection of E. coli serotype O157: H7 and Sh. dysenteriae type 1 strains was identified successfully using HRM curve analysis assay; it was employed for food samples as an accurate, specific and sensitive method for differentiating two pathogens

Summary

Introduction

The goal of this study was to develop HRMA PCR assay for differentiation and detection of stx1A gene to identify E. coli O157: H7 and Sh. dysenteriae type 1 isolated from food samples and determination of prevalence of these pathogens by this method. Specific primer pairs for identification of E. coli serotype O157: H7 and Sh. dysenteriae type 1 were designed based on detection of stx1A gene-encoded Shiga toxin released by these pathogens using IDT PrimerQuest online tool to design specific primers for qPCR intercalating dye reaction (https://eu.idtdna.com/Primerquest/ Home/Index) (IDT, USA).

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