RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

O’Hanlon, Karen; Catarame, T.M.G.; Duffy, Geraldine; Blair, I.S.; McDowell, D.A.

doi:10.1111/j.1365-2672.2004.02224.x

Cited by 44 publications

(19 citation statements)

References 18 publications

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“…As raw ground beef can be undercooked, E. coli O157:H7 was declared an adulterant (as defined in 9 CFR x301.2) in this product; (Anonymous, 1999;Donnenberg and Whittam, 2001) and there is interest in identifying the occurrence of other groups of species that elaborate these toxins (Karmali et al, 1985;Jaeger and Acheson, 2000;Bosilevac et al, 2007;Eblen, 2007) as they, too, have been shown to have public health impact (Neill et al, 1985;Murphy et al, 2005) and occur in beef (O'Brien et al, 1982;O'Hanlon et al, 2004).…”

Section: Introductionmentioning

confidence: 98%

Polymerase Chain Reaction Screening forSalmonellaand EnterohemorrhagicEscherichia colion Beef Products in Processing Establishments

Hill¹,

Suhalim²,

Richter³

et al. 2011

Foodborne Pathogens and Disease

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Pathogenic Escherichia coli strains on raw or insufficiently cooked foods are of public health concern as serious disease may result from their ingestion. Therefore, many commercial producers of beef products screen for E. coli O157:H7 before shipment. While Salmonella is not considered an adulterant on raw beef products, it is used as an indication of process control. To detect these microorganisms, rapid screening methods are often used to provide results within 8-24 hours after sampling. During 2005-2008, about 971,389 samples from several commercial beef production plants were tested using a rapid screening method based on the polymerase chain reaction to determine if they were presumptively positive for bacterial cells carrying Salmonella or Shiga toxin-producing E. coli-specific genes. Of the product lots sampled (trim, ground beef, and variety meats), 15% were positive for the stx(1) and/or stx(2) (Shiga toxin genes), 9.1% for the eae gene (the attaching and effacing gene [eae] encoding intimin), 3.0% for an rfb gene region (encoding the O157-specific O side chain polysaccharide), and 1.67% for Salmonella by the polymerase chain reaction assay. In general, lots of ground beef showed the lowest frequency of contamination, and variety meats (by-products of carcass evisceration), the highest. Overall, 4.6%, 4.6%, and 0.8% samples were screen-positive for enteropathogenic E. coli, enterohemorrhagic E. coli, and E. coli O157, respectively. Of the E. coli O157-positive samples, 14% were also Salmonella positive. The frequency of screen-positive samples increases during the summer months, probably because of the prevalence of climatic conditions more conducive to microbial growth. The presence of fecal organisms in beef products suggests a failure of sanitary controls during processing and the more prevalent relatives of E. coli O157, Shiga toxin-producing Escherichia coli, enteropathogenic E. coli, and enterohemorrhagic E. coli, serve as more sensitive indicators of contamination than O157 strains alone.

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Section: Introductionmentioning

confidence: 98%

Polymerase Chain Reaction Screening forSalmonellaand EnterohemorrhagicEscherichia colion Beef Products in Processing Establishments

Hill¹,

Suhalim²,

Richter³

et al. 2011

Foodborne Pathogens and Disease

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“…Hence, some false-negative results could be obtained after the IMS analysis, for which only 6 h of incubation is recommended (ISO EN 16654). For an enrichment with 6 h of incubation before detection by PCR, some false-negative results could be also obtained (20,34).…”

Section: Vol 72 2006mentioning

confidence: 99%

Modeling and Predicting the Simultaneous Growth of Escherichia coli O157:H7 and Ground Beef Background Microflora for Various Enrichment Protocols

Vimont

Vernozy-Rozand

Montet

et al. 2006

Appl Environ Microbiol

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The simultaneous growth of Escherichia coli O157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth and E. coli broth), the presence of novobiocin in the broth, and the incubation temperature (37°C or 40°C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40°C) did not inhibit BM growth, and incubation at 40°C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum-and non-modified-atmospherepacked ground beef.

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“…This is detrimental for reliable quantification. We demonstrated above that most of the primers and probes described in the literature for STEC detection or quantification [17,22,23,24,25,26,27,28,29,30,31] contained several mismatches when aligned to gene sequences of the different variants of stx1 , stx2 and eae . This implies that amplification is not optimal for some gene variants only.…”

Section: Discussionmentioning

confidence: 96%

“…Subtypes of stx were denominated according to the subtyping nomenclature established at the 7th International Symposium on Shiga Toxin (Verocytotoxin)-Producing Escherichia coli Infections (Buenos Aires, 10–13 May, 2009). Various primer-probe combinations for stx1 , stx2 and eae from the literature [17,22,23,24,25,26,27,28,29,30,31,32,33] and newly designed primers and probes using Primer Express 2.0 (Applied Biosystems, Foster City, CA, US), were aligned to the local database. The combinations that resulted in the fewest mismatches (≤2 in a primer, ≤1 in a probe) for the aforementioned subtypes (except stx2f ) were selected.…”

Section: Methodsmentioning

confidence: 99%

A qPCR Assay to Detect and Quantify Shiga Toxin-Producing E. coli (STEC) in Cattle and on Farms: A Potential Predictive Tool for STEC Culture-Positive Farms

Verstraete¹,

Coillie²,

Werbrouck³

et al. 2014

Toxins

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Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect and quantify Shiga toxin genes stx1 and stx2 and the intimin gene eae. Subsequently, 59 fecal samples from six farms were tested using qPCR and a culture method as a reference. Three farms had contaminated animals as demonstrated by the culture method. Culture-positive farms showed moderate significantly higher stx prevalences than culture-negative farms (p = 0.05). This is the first study which showed preliminary results that qPCR can predict STEC farm contamination, with a specificity of 77% and a sensitivity of 83%, as compared with the culture method. Furthermore, the presence or quantity of stx genes in feces was not correlated to the isolation of STEC from the individual animal. Quantitative data thus did not add value to the results. Finally, the detection of both stx and eae genes within the same fecal sample or farm using qPCR was not correlated with the isolation of an eae-harboring STEC strain from the respective sample or farm using the culture method.

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RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

Abstract: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.

Cited by 44 publications

References 18 publications

Polymerase Chain Reaction Screening forSalmonellaand EnterohemorrhagicEscherichia colion Beef Products in Processing Establishments

Polymerase Chain Reaction Screening forSalmonellaand EnterohemorrhagicEscherichia colion Beef Products in Processing Establishments

Modeling and Predicting the Simultaneous Growth of Escherichia coli O157:H7 and Ground Beef Background Microflora for Various Enrichment Protocols

A qPCR Assay to Detect and Quantify Shiga Toxin-Producing E. coli (STEC) in Cattle and on Farms: A Potential Predictive Tool for STEC Culture-Positive Farms

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