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

ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targetedeaesubtypes. The simultaneous presence ofstx,eae, and one of the five O group markers was found in 58.0% of the samples, and the five targetedstxpluseaeplus O genetic combinations were detected 143 times. However, taking into consideration the association betweeneaesubtypes and O group markers, the resultingstxpluseaesubtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22E. colistrains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive forstx,eaeand an O group marker, but that were negative for the correspondingeaesubtype, were successful. Characterization of the 24E. coliisolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenicE. coli(aEPEC). Finally, the more discriminatingeaesubtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

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Multiplex Real-Time PCR Assays for Screening of Shiga Toxin 1 and 2 Genes, Including All Known Subtypes, and Escherichia coli O26-, O111-, and O157-Specific Genes in Beef and Sprout Enrichment Cultures

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-050 ◽

2015 ◽

Vol 78 (10) ◽

pp. 1800-1811 ◽

Cited By ~ 6

Author(s):

TETSUYA HARADA ◽

ATSUSHI IGUCHI ◽

SUNAO IYODA ◽

KAZUKO SETO ◽

MASUMI TAGUCHI ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Screening Methods ◽

Direct Plating ◽

The Real ◽

Enrichment Cultures ◽

Quantitative Accuracy ◽

Pcr Assays

Shiga toxin family members have recently been classified using a new nomenclature into three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g). To develop screening methods for Stx genes, including all of these subtype genes, and Escherichia coli O26-, O111-, and O157-specific genes in laboratory investigations of Shiga toxin–producing E. coli (STEC) foodborne cases, we developed multiplex real-time PCR assays and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates, recombinant plasmids, and food enrichment cultures and by performing STEC spiking experiments with beef and sprout enrichment cultures. In addition, we evaluated the relationship between the recovery rates of the target strains by direct plating and immunomagnetic separation and the cycle threshold (CT) values of the real-time PCR assays for the Stx subtypes and STEC O26, O111, and O157 serogroups. All three stx1- and seven stx2-subtype genes were detected by real-time PCR with high sensitivity and specificity, and the quantitative accuracy of this assay was confirmed using control plasmids and STEC spiking experiments. The results of the STEC spiking experiments suggest that it is not routinely possible to isolate STEC from enrichment cultures with real-time PCR CT values greater than 30 by direct plating on MacConkey agar, although highly selective media and immunomagnetic beads were able to isolate the inoculated strains from the enrichment cultures. These data suggest that CT values obtained from the highly quantitative real-time PCR assays developed in this study provide useful information to develop effective isolation strategies for STEC from food samples. The real-time PCR assays developed here are expected to aid in investigations of infections or outbreaks caused by STEC harboring any of the stx-subtype genes in the new Stx nomenclature, as well as STEC O26, O111, and O157.

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DNA Extraction for Sensitive Detection of Shiga Toxin-Producing Escherichia coli in Food by Real-Time PCR Assays

Food Hygiene and Safety Science (Shokuhin Eiseigaku Zasshi) ◽

10.3358/shokueishi.60.183 ◽

2019 ◽

Vol 60 (6) ◽

pp. 183-186

Author(s):

Tetsuya Mori ◽

Sayaka Nagao-Sato ◽

Kanae Kishino ◽

Toyohiko Namba ◽

Yukiko Hara-Kudo

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Dna Extraction ◽

Shiga Toxin ◽

Real Time Pcr ◽

Sensitive Detection ◽

Pcr Assays

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Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

Microorganisms ◽

10.3390/microorganisms8111801 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1801

Author(s):

Michael Bording-Jorgensen ◽

Brendon D. Parsons ◽

Gillian A.M. Tarr ◽

Binal Shah-Gandhi ◽

Colin Lloyd ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Gene Detection ◽

Culture Positivity ◽

Hands On ◽

Culture Independent ◽

Chromogenic Agar ◽

Stool Samples

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

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Real-time PCR for differentiation of F18 variants among enterotoxigenic and Shiga toxin-producing Escherichia coli from piglets with diarrhoea and oedema disease

The Veterinary Journal ◽

10.1016/j.tvjl.2013.07.021 ◽

2013 ◽

Vol 198 (2) ◽

pp. 538-540 ◽

Cited By ~ 8

Author(s):

Jae-Won Byun ◽

Byeong Yeal Jung ◽

Ha-Young Kim ◽

John M. Fairbrother ◽

Myoung-Heon Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Oedema Disease

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Multiplex SYBR Green and duplex TaqMan real-time PCR assays for the detection of Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB

Molecular and Cellular Probes ◽

10.1016/j.mcp.2018.12.004 ◽

2019 ◽

Vol 43 ◽

pp. 20-28 ◽

Cited By ~ 2

Author(s):

Roberto Cruz-Flores ◽

Hung Nam Mai ◽

Arun K. Dhar

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Toxin Genes ◽

Pcr Assays

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Development of SYBR green-based real time PCR assays for detection and quantification of adulteration in wheat-based composite breads and their in-house validation

Journal of Cereal Science ◽

10.1016/j.jcs.2018.11.020 ◽

2019 ◽

Vol 85 ◽

pp. 91-97

Author(s):

Remziye Yılmaz ◽

Ceren Bayraç ◽

Arzu Başman ◽

Hamit Köksel

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Pcr Assays ◽

Detection And Quantification

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Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the South of Vietnam

10.21203/rs.3.rs-18430/v1 ◽

2020 ◽

Author(s):

Vu Thuy Duong ◽

Le Thi Phuong Tu ◽

Ha Thanh Tuyen ◽

Le Thi Quynh Nhi ◽

James I Campbell ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Middle Income ◽

Middle Income Countries ◽

Virulence Markers ◽

Low Middle Income Countries ◽

High Prevalence ◽

Pcr Assays

Abstract BackgroundDiarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. ResultsThe multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2,815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p<0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes. ConclusionsThis approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

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