title: Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.
abstract: Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.
authors.affiliation: 1 Animal and Veterinary Sciences Department, Center for Antimicrobial Research and Food Safety, California State Polytechnic University , Pomona, California.
authors.forename: Shelton E
authors.initials: SE
authors.lastname: Murinda
authors.forename: A Mark
authors.initials: AM
authors.lastname: Ibekwe
authors.forename: Syaizul
authors.initials: S
authors.lastname: Zulkaffly
authors.forename: Andrew
authors.initials: A
authors.lastname: Cruz
authors.forename: Stanley
authors.initials: S
authors.lastname: Park
authors.forename: Nur
authors.initials: N
authors.lastname: Razak
authors.forename: Farah Md
authors.initials: FM
authors.lastname: Paudzai
authors.forename: Liana
authors.initials: L
authors.lastname: Ab Samad
authors.forename: Khairul
authors.initials: K
authors.lastname: Baquir
authors.forename: Kokilah
authors.initials: K
authors.lastname: Muthaiyah
authors.forename: Brenna
authors.initials: B
authors.lastname: Santiago
authors.forename: Amirul
authors.initials: A
authors.lastname: Rusli
authors.forename: Sean
authors.initials: S
authors.lastname: Balkcom
mesh_terms: D015169:Colony Count, Microbial; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D045522:Recombinases; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2 / Q000235:genetics; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; D045522:Recombinases; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2