Novel Real-Time PCR Method To Detect Escherichia coli O157:H7 in Raw Milk Cheese and Raw Ground Meat

Abstract

Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.

Rapid Detection of Escherichia coli O157:H7 by Loop-Mediated Isothermal Amplification Coupled with a Lateral Flow Assay Targeting the z3276 Genetic Marker

“…The limit of detection of the z3276-LAMP-LF assay, as determined using artificially spiked ground beef and bovine feces, was 1.3 CFU/mL after overnight enrichment (generally > 12 h), compared with 10 CFU/mL after 4-h enrichment by the z3276-LAMP assay using turbidity observations (Ravan et al 2016) and 80 CFU/g for spiked beef with an 8-h enrichment by z3276-real-time PCR detection (Li and Chen 2012). Besides the z3276 gene, other target genes have been used to develop various detection methodologies, i.e., a real-time PCR targeting uidA showed 10 2 CFU/mL sensitivity after 24-h enrichment (Miszczycha et al 2012). When the z3276-LAMP-LF assay was applied to detect E. coli O157:H7 from slaughterhouse samples, it also presented superiority to conventional duplex PCR methods (Li et al 2011) due to its higher positive detection rate (7.9% vs. 7.0%) and simpler operation.…”

Use of ALLGIO Probe Assays for Detection of HBV Resistance to Adefovir in Patients with Chronic Hepatitis B, Kerman, Iran

“…This leads to the separation of the two reporter dyes and de-quench occurs. As this process releases two fluorescing molecules instead of one, the signal change is much greater, making AllGlo-based quantitative PCR detections highly sensitive and robust (Miszczycha et al, 2012). Since AllGlo requires only single dye component, it greatly simplifies the manufacturing process.…”

Short communication: Behavior of different Shiga toxin-producing Escherichia coli serotypes (O26:H11, O103:H2, O145:H28, O157:H7) during the manufacture, ripening, and storage of a white mold cheese

“…The identification and characterization of the target bacteria were performed by real-time PCR as previously described (Nielsen and Andersen, 2003;Perelle et al, 2004Perelle et al, , 2005Madic et al, 2010;Miszczycha et al, 2012). The serotype effect was tested.…”