A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR

McKillip, John L.; Jaykus, Lee‐Ann; Drake, M.A.

doi:10.1046/j.1365-2672.2000.01079.x

Cited by 42 publications

(31 citation statements)

References 26 publications

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“…Most methods described in the literature involve prior separation of the cells (Allmann et al, 1995;Herman et al, 1997;Serpe et al, 1999;Torriani et al, 1999;McKillip et al, 2000;Coppola et al, 2001;Ogier et al, 2002;Randazzo et al, 2002;Ercolini et al, 2003;Furet et al, 2004;Ogier et al, 2004;Baruzzi et al, 2005;Rudi et al, 2005;Rademaker et al, 2006;ElBaradei et al, 2007;Lopez-Enriquez et al, 2007;Parayre et al, 2007;Rossmanith et al, 2007;Trmcic et al, 2008;Van Hoorde et al, 2008;Alegría et al, 2009;Dolci et al, 2009;Zago et al, 2009;Le Dréan et al, 2010;Mounier et al, 2010). The recovery of cells from milks or fermented milks is easier to perform than from cheeses.…”

Section: Dna Extractionmentioning

confidence: 99%

“…In direct DNA extraction procedures (McKillip et al, 2000;Duthoit et al, 2003;Feurer et al, 2004a;Feurer et al, 2004b;Callon et al, 2006;Monnet et al, 2006;Delbes et al, 2007;Masoud et al, 2011), the cheese samples are first homogenised in a liquid solution by a method involving bead-beating, a mortar and pestle or other mechanical treatments. Efficient treatments of casein degradation and cell lysis, followed by phenol/chloroform extractions, are then needed to remove most contaminating compounds.…”

Section: Dna Extractionmentioning

confidence: 99%

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Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

Monnet¹,

Matijašić²

2012

Polymerase Chain Reaction

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Section: Dna Extractionmentioning

confidence: 99%

Section: Dna Extractionmentioning

confidence: 99%

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

Monnet¹,

Matijašić²

2012

Polymerase Chain Reaction

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“…With RT-PCR, matrix effects between broth and meat enrichments were more pronounced and more variable. Decreases in cell and/or nucleic extraction efficiency from the meat enrichment compared to broth are a likely source of the decreased sensitivity by both DNA and mRNA from meat enrichments (18,19). The differences in sensitivity of detection of mRNA compared to DNA may represent the inherent instability of mRNA compared to DNA or inhibition of the reverse transcriptase step of RT-PCR.…”

Section: Vol 68 2002mentioning

confidence: 99%

“…b TSB, tryptic soy broth; ME, modified E. coli enrichment broth with added novobiocin; EEB, E. coli enrichment broth. (5,11,19). The use of bacterial mRNA as a means of pathogen detection in foods, particularly that of slt mRNA of E. coli O157:H7, has not been widely studied.…”

Section: ϫ4mentioning

confidence: 99%

Optimization of Reverse Transcriptase PCR To Detect Viable Shiga-Toxin-Producing Escherichia coli

McINGVALE

Elhanafi

Drake

2002

Appl Environ Microbiol

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The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing Escherichia coli (STEC) was investigated. Four primer sets, each targeting a specific region in the slt-II operon, were evaluated for their stringency and specificity for slt-II mRNA. STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration. Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments. Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h. RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots. mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g. These results indicate that RT-PCR of E. coli slt-II mRNA is useful for detection of viable STEC in ground beef.Escherichia coli O157:H7 was first isolated as a human pathogen in 1982 and has since become a well-known agent of food-borne illness. Cases of hemorrhagic colitis, hemolytic uremic syndrome, and death have been reported following the consumption of raw or undercooked ground beef (8). Although most commonly associated with foods of animal origin, enterohemorrhagic E. coli (EHEC) may also be isolated from contaminated drinking water and acidic foods such as mayonnaise, salad dressings, and buttermilk (6,16,29). The infectious dose for E. coli O157:H7 and other serotypes of EHEC varies greatly between populations. For susceptible individuals such as the elderly and infants, the infectious dose may be as low as 10 cells (8).Without an animal model, there is much controversy over which virulence factors of EHEC contribute to human pathogenicity (8). EHEC produce Shiga-like toxins (SLTs) that characterize this group of pathogenic E. coli. Other virulence markers include intimin, hemolysin, and the locus of enterocyte effacement (8). Food-borne illnesses have occurred with isolates that possess all or only a few of these markers (8, 9). Possession of slt genes is a consistent virulence marker (1), but without evidence of human illness, E. coli possessing slt are often referred to as Shiga toxin-producing E. coli (STEC). Despite the fact that EHEC strains containing slt-I and slt-II have been isolated from patients with hemorrhagic colitis, studies have shown that strains possessing only slt-II are more frequently associated with human disease complications (8, 25).Molecular methods for the detection of E. coli O157:H7 are becoming more widely accepted as an alternative to traditional growth-based tests. The ability to specifically identify the presence of an organism within a fraction of the time required by standard approaches makes molecular tests ideal for use in food systems (2). A number of recent reports have applied molecular tools for specific detection of pathogenic E. coli in food systems (10,24,31,32). Most of these assays have used DNA as the target molecule; however, recent literature has sh...

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“…Cells were enumerated by pour plate counts of TSB with violet red bile (Difco) overlay incubated at 32°C for 24 h. Prior to the use of pasteurized skim milk in assays, plate counts of TSB with violet red file overlays were conducted with the milk to confirm that it was free of coliforms. Cells were concentrated from broth or milk, and DNA was extracted using the method described by McKillip et al (7).…”

mentioning

confidence: 99%

Development of a Quantitative Competitive PCR Assay for Detection and Quantification of Escherichia coli O157:H7 Cells

Drake

2001

Appl Environ Microbiol

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A quantitative competitive PCR (QC-PCR) assay was developed to detect and quantify Escherichia coli O157:H7 cells. From 10 3 to 10 8 CFU of E. coli O157:H7 cells/ml was quantified in broth or skim milk, and cell densities predicted by QC-PCR were highly related to viable cell counts (r 2 ‫؍‬ 0.99 and 0.93, respectively). QC-PCR has potential for quantitative detection of pathogenic bacteria in foods.

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A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR

Cited by 42 publications

References 26 publications

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

Optimization of Reverse Transcriptase PCR To Detect Viable Shiga-Toxin-Producing Escherichia coli

Development of a Quantitative Competitive PCR Assay for Detection and Quantification of Escherichia coli O157:H7 Cells

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