mericon E. coli O157 Detection Kits

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).

The mericon E. coli STEC O-Type Kit exhibits high specificity for E. coli strain O157:H7, O26, O45, O103, O111, O121, and O145. The kit was tested against an extensive inclusivity panel of E. coli O157:H7 and non-O157:H7 serotype strains, and an exclusivity panel of other E. coli and non-E. coli strains. All tested O157:H7 and non-O157:H7 serotype strains (O26, O45, O103, O111, O121, and O145) were successfully detected and no cross-reactivity was observed with other pathogens using 2500 copies of tested DNA (see table "Results of mericon E. coli STEC O-Type Kit cross-reactivity study").

Results of mericon E. coli O157 Screen Plus Kit cross-reactivity study

Pathogen/gene	Result	Pathogen/gene	Result
eae	+	Enterobacter sakazakii	–
E. coli O157:H7	+	Escherichia coli O6	–
VTEC stx1	+	Listeria monocytogenes	–
VTEC stx2	+	Shigella flexneri	–
Bacillus cereus	–	Staphylococcus aureus	–
Camplyobacter jejuni	–	Yersinia enterocolitica	–
Clostridium perfringens	–

Results of mericon E. coli STEC O-Type Kit cross-reactivity study

Pathogen	Strain no.	Result Orange channel	Result Green channel
E. coli O26:H–	DSM 8695	–	+
E. coli O45	SSI 81886	–	+
E. coli O103	NC09103	–	+
E. coli O103	SSI 82109	–	+
E. coli O111:H–	DSM 8698	–	+
E. coli O111:H–	DSM 10234	–	+
E. coli O121	NC09121	–	+
E. coli O145	SSI 82280	–	+
E. coli O157:H7	DSM 8579	+	–
E. coli O157:H7	DSM 13526	+	–
E. coli O157:H7	DSM 17076	+	–
E. coli O157:H7	DSM 19206	+	–
E. coli O1:H7	DSM 30083	–	–
E. coli O1:K1:H7	ATCC11775	–	–
E. coli O1:K1:H7	DSM 10750	–	–
E. coli O2:K1:H4	DSM 10777	–	–
E. coli O7:K1:H7	DSM 10858	–	–
E. coli O11:H10	DSM 11751	–	–
E. coli O12:H2	DSM 8703	–	–
E. coli O13:H11	DSM 1058	–	–
E. coli O18ac:K1:H7	DSM 10723	–	–
E. coli O19:H7	DSM 11752	–	–
E. coli O25:H4	DSM 22664	–	–
E. coli O29:H10	DSM 9026	–	–
E. coli O104	NC09104	–	–
E. coli O107:H5	DSM 9033	–	–
E. coli O112ac:H–	DSM 9027	–	–
E. coli O113	SSI 82121	–	–
E. coli O124:H30	DSM 9031	–	–
E. coli O126:H2	DSM 8701	–	–
E. coli O143:H–	DSM 9028	–	–
E. coli RM-HYG118	VTEC isolate	–	–
E. coli RM-HYG119	VTEC isolate	–	–
E. coli RM-HYG120	VTEC isolate	–	–
Bacillus cereus	ATCC 11778	–	–
Campylobacter jejuni	ATCC 33560	–	–
Clostridium perfringens	ATCC 13124	–	–
Enterobacter sakazakii	DSM 4485	–	–
Escherichia coli	ATCC	–	–
Escherichia coli	NCTC 9002	–	–
Legionella erythra	Sifin	–	–
Listeria monocytogenes	ATCC 7644	–	–
Shigella flexneri	ATCC 12022	–	–
Staphylococcus aureus	ATCC 25923	–	–
Vibrio vulnificus	ATCC 43478	–	–
Yersinia enterocolitica	ATCC 9610	–	–

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").

Interpretation of results with the mericon E. coli O157 Screen Plus Kit

No.	Orange O157	Green stx1/stx2	Crimson eae	Yellow IC	Next action
1	–	–	–	Valid	No further action
2	–	+	–	Valid	No further action
3	–	–	+	Valid	No further action
4	+	–	–	Valid	Additional testing required
5	–	+	+	Valid	Additional testing required
6	+	+	+	Valid	Additional testing required
7	–	–	–	Invalid	Dilute sample and repeat test

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 ”).

Interpretation of results with the mericon E. coli STEC O-Type Kit

No.	O157:H7 Orange channel	Non-O157:H7 serotypes Green channel	IC Yellow channel	Next action
1	–	–	Valid	No further action
2	–	+	Valid	Additional testing required
3	+	–	Valid	Additional testing required
4	+	+	Valid	Additional testing required
5	–	–	Invalid	Dilute sample and repeat test

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.

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.

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.