A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7

Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

doi:10.1016/j.micpath.2015.12.011

Cited by 46 publications

(36 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…,b; Ravan et al . ). However, to the best of our knowledge, no LAMP assays able to detect an E. coli ‐specific gene and verotoxin producing genes simultaneously, as well as distinguish between generic E. coli and VTEC, are currently available.…”

Section: Introductionmentioning

confidence: 97%

A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenicEscherichia coliin beef and bovine faeces

et al. 2017

View full text Add to dashboard Cite

show abstract

Section: Introductionmentioning

confidence: 97%

A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenicEscherichia coliin beef and bovine faeces

et al. 2017

View full text Add to dashboard Cite

show abstract

“…As most of the conventional methods require at least 2-3 days for getting the result and another one week for confi rmation, PCR-based methods that target some specifi c virulence factor genes such as stx1, stx2, eaeA, fl iC, Z3276 and hlyA, are used for detection of E.coli O157:H7 strains [Aydin et al, 2014;Ravan et al, 2016]. Some previously published reports from our lab and elsewhere A large number of fi eld applications showed that the O157 immunomagnetic method greatly improved the isolation rate of bacteria [Buesa et al, 2002;Wolf et al, 2007].…”

Section: Introductionmentioning

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.

View full text Add to dashboard Cite

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

show abstract

“…An enrichment Rti‐LAMP (E‐Rti‐LAMP) assay targeting Z3276 gene of E. coli O157:H7 was also established and had similar sensitivity with that of VT2 gene (data not shown). It has been reported that a LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10 4 CFU/g without enrichment and 10 2 CFU/g after a 4 hr pre‐enrichment (Ravan et al, ). Wang, Jiang, Yang, Prinyawiwatkul, and Ge () also reported that LAMP could detect as low as E. coli 10–20 CFU per 25 g in beef before an 8 hr pre‐enrichment.…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, Vero toxins (VT) which divided into VT1 and VT2 are the main pathogenic factors of E. coli O157:H7, and some strains of E. coli O157 produced only VT2, other O157 strains produced both VT1 and VT2 (Ludwig, Karmali, Smith, & Petric, ; Scotland, Willshaw, Smith, & Rowe, ; Willshaw, Smith, Scotland, Field, & Rowe, ). Z3276 gene is a unique genetic marker for the identification of E. coli O157:H7 strains, which showed a high sequence identity and distinguished E. coli O157:H7 from other bacteria for its specific attachment to a particular tissue or surface (Ravan, Amandadi, & Sanadgol, ). Therefore, the VT2 gene and Z3276 gene were selected as real‐time loop‐mediated isothermal amplification (Rti‐LAMP) primer for the detection of E. coli O157:H7 in this study.…”

Section: Introductionmentioning

confidence: 99%

Real‐time loop‐mediated isothermal amplification assays combined with ethidium monoazide bromide and bentonite coated activated carbon for rapid and sensitive detection of viable Escherichia coli O157:H7 from milk without enrichment

Zhang

Zhong

Shu

et al. 2019

Journal of Food Safety

View full text Add to dashboard Cite

A real‐time loop‐mediated isothermal amplification (Rti‐LAMP) DNA assay was developed for rapid and sensitive detection of viable Escherichia coli O157:H7 in milk by combining with ethidium monoazide bromide (EMA) and bentonite coated activated carbon (BCAC) treatment without enrichment. The assay involved inoculating 25 mL portions of milk with various numbers of E. coli O157:H7. Then each of milk sample was adsorbed with 4 g BCAC to remove DNA amplification inhibitor. The recovered purified bacteria sample was further treated with EMA for discrimination viable from dead cells in Rti‐LAMP assay. The detection limit of viable E. coli O157:H7 was 10 CFU/mL in milk by the BCAC‐EMA‐Rti‐LAMP, and the entire assay could be completed in 5 hr. Twenty‐five raw milk samples were detected by the BCAC‐EMA‐Rti‐LAMP assay, using three methods as controls including E. coli O157:H7 plate culture, BCAC‐Rti‐LAMP, and Rti‐LAMP combined with enrichment at 37°C (E‐Rti‐LAMP). The results showed that the BCAC‐EMA‐Rti‐LAMP had similar accuracy and sensitivity as that of plate culture, as they both detected one sample of viable E. coli O157:H7 positive from 25 fresh raw milk samples. These data strongly suggested that the BCAC‐EMA‐Rti‐LAMP could rapidly and sensitively detect viable E. coli O157:H7 in milk without enrichment. Practical Applications Escherichia coli O157:H7 is one of the foodborne pathogens in milk, which has low dose of infection and high pathogenicity. In milk, components such as Ca2+, proteinase, fats, and milk proteins may block DNA and shield it from access by polymerase. This study was targeting VT2 and Z3276 genes for rapid and sensitive detection of viable E. coli O157:H7 in milk. BCAC was used to remove DNA amplification inhibitor and improve the detection sensitivity. EMA was used to discriminate viable from dead cells. The assay will be a potential tool in the rapidity, sensitivity, and specificity for detection of E. coli O157:H7 in milk without enrichment.

show abstract

A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7

Cited by 46 publications

References 34 publications

A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenicEscherichia coliin beef and bovine faeces

A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenicEscherichia coliin beef and bovine faeces

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

Real‐time loop‐mediated isothermal amplification assays combined with ethidium monoazide bromide and bentonite coated activated carbon for rapid and sensitive detection of viable Escherichia coli O157:H7 from milk without enrichment

Contact Info

Product

Resources

About