mericon E. coli O157 Detection Kits
For real-time PCR screening for E. coli serotype O157 and detection of E. coli serotype O157:H7 in food/animal feed
The mericon E. coli O157 Screen Plus Kit is designed for the qualitative detection of Escherichia coli serotype O157 and E. coli virulence genes intimin (eae) and Shiga toxin-like proteins (stx1 and stx2). The presence of eae and stx1/stx2 suggests that further investigation into the E. coli serotype O157, or the non-O157 O-serotypes (O26, O45, O103, O111, O121, and O145) is required in food and animal feed samples after enrichment. This can be carried out using the mericon E. coli STEC O-Type Kit.
mericon E. coli O157 Detection Kits are intended for molecular biology applications in food, animal feed, and pharmaceutical product testing. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Performance
The mericon E. coli Screen Plus Kit is designed for the qualitative detection of the E. coli serotype O157 and E. coli virulence genes intimin (eae) and Shiga toxin-like proteins (stx1 and stx2). The presence of eae and stx1/stx2 suggests that further investigation into the E. coli O-serotype O157, or the non-O157 O-serotypes (O26, O45, O103, O111, O121, and O145) is required in food and animal feed samples after enrichment. The assay can detect as few as 10 copies of E. coli DNA in a reaction and it exhibits high specificity for pathogens of this species. In an experiment using 2500 copies of the tested DNA, no cross-reactivity was observed with other pathogens (see table "Results of mericon E. coli O157 Screen Plus Kit cross-reactivity study" for exclusivity/inclusivity data).
Principle
According to national regulations such as the USDA FSIS regulations for the US beef industry, all non-intact products (beef trimmings) are required to be tested, or should be tested, for 6 strains of shiga toxigenic E. coli (STEC) — the "big 6" — before they can be shipped to the food distribution chain. The mericon E. coli testing workflow provides a complete STEC screening and confirmation solution for a variety of food products. The assays are run on QIAGEN Rotor-Gene Q real-time PCR instruments able to detect at least 4 channels: Rotor-Gene Q 5plex, Rotor-Gene Q 5plex HRM, and Rotor-Gene Q 6plex. The workflow detects the "big 6" STECs, as well as E. coli O157:H7. The first step of the workflow — screening for potentially positive samples — is carried out using the mericon E. coli O157 Screen Plus Kit. The second step of the workflow — identification of E. coli O157:H7 and distinction from other non-O157 O-type strains — is carried out using the mericon E. coli STEC O-Type Kit. The mericon E. coli O157 Screen Plus Kit is designed for the qualitative detection of the E. coli serotype O157 and E. coli virulence genes intimin (eae) and Shiga toxin-like proteins (stx1 and stx2). The presence of eae and stx1/stx2 suggests that further investigation into the presence of E. coli O-serotype O157, or the non-O157 O-serotypes (O26, O45, O103, O111, O121, and O145) is required in food and animal feed after enrichment. The mericon E. coli O157 Screen Plus Kit is designed to allow easy decision making in the next step of the workflow and interpretation can be summarized by a simple table (see the table "Interpretation of results with the mericon E. coli O157 Screen Plus Kit").
After further testing is carried out using the mericon E. coli STEC O-Type Kit, the results allow easy interpretation of final identification results that can be summarized in a simple table ( see the table “Interpretation of results with the mericon E. coli STEC O-Type Kit ”).
Detection of pathogens using real-time PCR is based on the amplification of a specific region of the relevant pathogen genome. The amplified product is detected using target-specific fluorescent probes that bind to it. As the PCR product accumulates, there is an increased fluorescent signal from the bound probes. Monitoring the fluorescence intensities during the PCR run (i.e., in real time) allows the detection of accumulating PCR product without having to reopen the reaction tubes afterwards. mericon PCR assays include an internal control to control for potential inhibition of the PCR reaction. Presence of the internal control aids in the straightforward interpretation of results (see tables "Interpretation of results with the mericon E. coli O157 Screen Plus Kit" and “Interpretation of results with the mericon E. coli STEC O-Type Kit"). The PCR primer set for each assay is highly specific and targets a unique and conserved region of the tested pathogen genome that has been verified bioinformatically and experimentally.
Procedure
DNA is extracted from an enrichment culture of a sample of food or animal feed using a dedicated sample preparation kit. The mericon Bacteria Kit or the QIAsymphony mericon Bacteria Kit are recommended for the isolation of bacterial DNA from Gram-negative bacteria. The isolated, pure bacterial DNA is assayed using a streamlined real-time PCR protocol that delivers results in 73 minutes on QIAGEN's Rotor-Gene Q. Results are displayed as easy-to-interpret PCR curves.
Applications
The mericon E. coli testing workflow provides a complete STEC screening and confirmation solution for a variety of food products. The assays are run on QIAGEN Rotor-Gene Q real-time PCR instruments able to detect at least 4 channels: Rotor-Gene Q 5plex, Rotor-Gene Q 5plex HRM, and Rotor-Gene Q 6plex. The workflow detects the "big 6" STECs, as well as E. coli O157:H7. The first step of the workflow — screening for potentially positive samples — is carried out using the mericon E. coli O157 Screen Plus Kit. The second step of the workflow — identification of the E. coli O157:H7 and distinction from other non-O157 O-type strains — is carried out using the mericon E. coli STEC O-Type Kit.
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