E. coli O157:H7 / O157 qPCR Kit

Escherichia coli (E. coli) is a bacterium that is commonly found in the gut of humans and warm-blooded animals and most of the strains are harmless. Some strains, however, such as enterohemorrhagic E. coli (EHEC) O157:H7, can cause serious foodborne outbreaks that cause diarrhoea, fever, and vomiting in humans. E. coli O157:H7 is recognized by its somatic (cell wall) antigen (O157) and its flagella antigen (H7). In addition, E. coli O157:H7 is known to produce Shiga-like toxins, which cause severe symptoms.

The reservoir of this pathogen appears to be mainly cattle, in addition to others such as ruminants, mammals, and birds.

E. coli O157:H7 is transmitted to humans primarily through the consumption of contaminated foods, such as raw or undercooked ground meat products and raw milk. Fecal contamination of water and other foods and cross-contamination during food preparation (with beef and other meat products, contaminated surfaces, and kitchen utensils) will also lead to infection.

The kit includes an Internal Control (IC). The IC is used for the evaluation of PCR inhibitors in the sample, or for the evaluation of problems that occurred during PCR preparation/amplification. The kit includes Master mix for the target and IC, primers and TaqMan probes, labelled with non-fluorescent quenchers. The signal for the detection of the targets is detected in the FAM (E. coli O157) and HEX/VIC (serotype H7) channels, and the one for the IC in the ROX channel. The kit contains Uracil-DNA Glycosylase (UDG), preventing DNA contamination with PCR products. Included in the kit is also a positive control, allowing an evaluation of the primers and probes used for the detection of the targets, and a negative control (nuclease-free water) to confirm the integrity of the kit reagents.

The kit was validated in the instrument ABI PRISM 7500 Fast. The kit is compatible with all thermocyclers working in FAM, JOE/VIC/HEX and ROX channels. The detection kit must not be used for diagnostic procedures. For food use only.

1. Sample enrichment
2. DNA extraction
3. PCR set-up and run
4. Results and interpretation

In accordance with ISO 9001, each lot of the kit is tested against predetermined specifications to ensure consistent product quality.

Molecular biology procedures, such as DNA extractions and PCR amplification, require qualified staff to prevent the risk of erroneous results, especially due to sample contamination or degradation of the nucleic acids contained in the samples. It is strongly recommended to have dedicated areas, materials and equipment for the DNA extraction, preparation of the PCR and post-PCR procedures. Workflow in the laboratory must proceed in a unidirectional manner, beginning in the Extraction Area and moving to the Amplification and Detection Area.

The user should always pay attention to the following:

• Read all the instructions provided before running the assay.
• Do not mix reagents from different batches.
• Wear proper PPE, including disposable gloves and laboratory coats.
• Store and extract positive material separately from all other reagents.

The kit should be stored between -20 °C and 5 °C and protected from exposure to light. Repeated thawing and freezing (> 5) should be avoided, as this may reduce the sensitivity. Consider freezing in aliquots to maintain the performance of the assay.

The kit contains reagents for 100 reactions.

• Microcentrifuge
• Laminar air flow cabinets/PCR cabinets
• Disposable powder-free gloves
• Micropipettes and nuclease-free filter tips
• Real-time PCR instrument
• Tubes/strips/multiwell plates and accessories specific for each instrument
• Lysis buffer / DNA extraction kit

Enrichment / DNA extraction

A pre-enrichment according to ISO/TS 13136, or E. coli isolated colonies, is recommended.

DNA extraction

Use a kit or suitable protocol for DNA extraction, such as Complex Extraction Kit (LC-6500), Extraction Rapid (LC-6504, LC-6505) or Extraction Column (LC-6506, LC-6507).

PCR preparation: PCR mix

Always wear gloves for all PCR procedures.

1. Thaw the kit solutions. Mix thoroughly (do not vortex the Enzymes tube) and centrifuge briefly.
2. Prepare the reactions, as described in the table below.
3. Mix the prepared Mix by inverting the tube and centrifuge briefly.
4. Dispense 15 µL aliquots of prepared Mix into the plate wells or PCR tubes.
5. For the negative control, pipette 5 µL of the Negative Control tube (clear cap); pipette 5 µL of DNA sample per well; and for the positive control, pipette 5 µL of the Positive Control tube (red cap). Each PCR tube/well should have a final PCR volume of 20 µL.
6. Centrifuge briefly the plate wells or PCR tubes.
7. Place the reactions into the real-time PCR instrument.

Note: Prepare the PCR reaction for each sample or, in the alternative, prepare a Master Mix for the total number of reactions plus 10% (e.g. for 10 samples, prepare a volume for 11). In this case, prepare the Mix in a 1.5 mL sterile, nuclease-free tube. Include 2 PCR reactions for the Positive and Negative controls.

PCR preparation: program set up

Prepare the real-time PCR instrument according to the following temperature/time program:

For analysis of PCR results, select fluorescence display options. Samples with positive Ct values are considered positive.

Important: Please also check amplification curves, not only Ct values. Samples should be inspected both in logarithmic and linear scale view and compared with the negative control. Adjust the threshold, if necessary. Sample results should be assessed after the positive and negative controls have been examined and determined to be valid. If the results of controls are not valid, the sample results cannot be interpreted.

Interpretation: controls

To validate the assay, the controls must have the following results:

Note that if the controls do not match these results, the experiment must be repeated.

Interpretation: samples

Interpretation of sample results is summarized in the following table:

(1) When both E. coli and IC detection are negative, this means the presence of PCR inhibitors in the sample. Dilute the sample or perform another DNA extraction.

(2) High DNA concentration of the target in the sample can lead to a reduced or absent fluorescence signal of the IC.

1. 100% exclusivity, determined using 13 strains of closely related organisms or occurring in the same habitat, including non-pathogenic E. coli and other serotypes of E. coli (Table 1).
2. 100% inclusivity, determined in 9 strains of E. coli O157:H7 and E. coli O157 (Table 1).

Table 1: Exclusivity and inclusivity of the kit

A detection limit of 1 to 10 cells per 25 g of food sample can be achieved after enrichment. The kit has a reaction sensitivity of 1 pg of target DNA.