A comparison of immunomagnetic separation and culture, RevealTM and VIPTM for the detection of E. coli O157 in enrichment cultures of naturally-contaminated raw beef, lamb and mixed meat products

Chapman, P. A.; Ellin, M.; Ashton, R.

doi:10.1046/j.1472-765x.2001.00883.x

Cited by 32 publications

(23 citation statements)

References 28 publications

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“…EHEC O157:H7 is clinically important due to its high virulence, extremely low infectious dose, and its ability to cause serious illness after a short course of acute infection. Identifi cation of food source with traditional detection methods based on isolation by bacterial culture and confi rmation with biotyping and serotyping is unreliable, as it is more time consuming and often shows low sensitivity in recovering small numbers from the samples [Chapman et al, 2001]. As an emerging foodborne bacterial pathogen, there is an urgent need to develop simple, rapid, sensitive and specifi c detection methods to ensure food safety, effective prevention and control of EHEC O157:H7 spread.…”

Section: Discussionmentioning

confidence: 99%

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Qin¹,

Puthiyakunnon²,

Zhang³

et al. 2018

Pol. J. Food Nutr. Sci.

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Escherichia coli O157:H7 is well known for many foodborne outbreaks that lead to fatal infections in human being worldwide. The objective of this study was to develop a rapid and sensitive method for detection of EHEC O157:H7 from ground beef using a method that combined immunomagnetic separation (IMS) with loop-mediated isothermal amplifi cation (LAMP). The EHEC O157:H7 cells were separated with Dynabeads coated with anti-EHEC O157:H7 after a short enrichment for 4 h. Then, EHEC O157:H7 was identifi ed by LAMP assay for amplifying and detecting the rfbE gene, which is highly conserved in all EHEC O157:H7 strains and exhibits strain-specifi c gene expression. The LAMP method results analyzed with real time turbidity measurements showed a high specifi city and sensitivity, with a positive detection rate of amplifi cation of EHEC O157:H7 DNA diluted to a minimum equivalent concentration of 1.8 × 10 1 CFU/mL, which was 10 times more sensitive than the conventional PCR assay. The IMS followed with LAMP could capture and detect a bacterial concentration as low as 3×10 1 CFU/mL from the meat samples, which was close to the sensitivity of LAMP assay with pure culture. IMS combined with realistic LAMP method is a simple, rapid, highly specifi c gene amplifi cation technology that is suitable for implementing as a screening assay in basic laboratory and fi eld test for detecting food contamination. Unauthenticated Download Date | 5/11/18 5:54 AM

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Section: Discussionmentioning

confidence: 99%

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Qin¹,

Puthiyakunnon²,

Zhang³

et al. 2018

Pol. J. Food Nutr. Sci.

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“…Thus, the early detection of VT is important to save both time and cost. The present culture methods require more than 3 days, and rapid detection methods like enzyme immunoassay require a high population of the target pathogen (Bennett et al, 1996;Chapman et al, 2001). A PCR assay was developed to rapidly detect target pathogens in enrichment cultures; however, the assay generally requires electrophoresis to detect the amplicon, which takes several hours.…”

Section: Introductionmentioning

confidence: 99%

Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification

Hara-Kudo

Nemoto

Ohtsuka³

et al. 2007

Journal of Medical Microbiology

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A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing Escherichia coli rapidly (within 60 min). The 24 strains of VT-producing E. coli were successfully amplified, but 6 strains of non-VT-producing E. coli and 46 bacterial species other than E. coli were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test using serogroups O157, O26 and O111 of VT-producing E. coli; this sensitivity is greater than that obtained by PCR assay. Furthermore, the LAMP assay was examined for its ability to detect VT-producing E. coli in food because of the difficulty of detection in food samples. The recovery of VT-producing E. coli by LAMP assay from beef and radish sprouts inoculated with the pathogen was high, similar to that obtained using culture methods with direct plating and/or plating after immunomagnetic separation. Although PCR assay was unable to recover VT-producing E. coli from half of the radish samples, LAMP assay was successful in most samples. In addition, VT-producing E. coli was successfully detected in cultures of the beef samples by LAMP assay, but not by the culture method. The LAMP products in naturally contaminated beef samples were analysed to confirm the specific amplification of the VT-encoding gene, and were found to show a specific ladder band pattern on agarose gel after electrophoresis. Additionally the sequences of the LAMP products coincided well with the expected sequences of the VT-encoding gene. These results indicate that the proposed LAMP assay is a rapid, specific and sensitive method of detecting the VT-producing E. coli.

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“…It is well recognized that E. coli O157:H7 has a bovine reservoir and that the organism is well adapted for life in the ruminant gastrointestinal tract (6,7,14,32). In contrast to humans, cattle colonized by E. coli O157:H7 are asymptomatic.…”

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confidence: 99%

Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains

Zhang

Laing

Zhang

et al. 2010

Appl Environ Microbiol

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Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx 2 flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx 2 ) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx 2 , whereas all LII strains carried variant stx 2c and 4 of 14 LI/II strains had copies of both stx 2 and variant stx 2c . Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/IIstrains produce significantly more stx 2 mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

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A comparison of immunomagnetic separation and culture, RevealTM and VIPTM for the detection of E. coli O157 in enrichment cultures of naturally-contaminated raw beef, lamb and mixed meat products

Cited by 32 publications

References 28 publications

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification

Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains

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