GLISA-Rapid Test (Gold Labelled ImmunoSorbent Assay) for the qualitative detection of E.coli O157 in food.
AOAC Approved.
Introduction
Amongst the E.coli human pathogens, Verotoxin (a Shiga like toxin) - forming strains (VTEC) have gained in importance in recent years. The group of enterohaemorrhagic E.coli (EHEC) with its highly pathogenic serovars O157:H7 strain is particularly interesting in this respect. EHEC are capable of initiating life-threatening illnesses, particularly in those with immune deficiency , young children and the elderly. The main sources of infection are contaminated, raw or insufficiently heated foods of animal origin, e.g. meat and dairy products. The reservoir for EHEC is the faeces of the cattle, sheep and goats. These microorganisms can enter food during the processing of meat and dairy products if hygienic conditions are inadequate.
The drastic increase in the incidence of food infection caused by E.coli O157 demands reliable and rapid methods of detection. Apart from traditional culture methods, immunological techniques are becoming more and more popular with users due to their better specificity and sensitivity.
Singlepath®E.coli O157 is an immunological screening test based on the immune flow principle and is designed in such a way that time-consuming and personnel intensive working steps are avoided.results.
Principle
Singlepath®E.coli O157 ( 1.04141) is an immunochromato- graphic rapid test based on gold-labelled antibodies. The test device has a circular sample port, and an oval shaped test (T) and control (C) window.
1. The sample is applied to the chromatography paper via the circular sample port.
2. The sample is absorbed through the pad to the reaction zone containing colloidal, gold-labelled antibodies specific to E.coli O157.
3. Any E.coli O157 antigen present complexes with the gold-labelled antibody and migrates through the port until it encounters a binding zone in the test (T) area.
4. The binding zone (T) contains another anti- E.coli O157
Antibody, which immobilizes any E.coli O157 -antibody complex present. Due to
the gold-labelling, a distinct red line is then formed.
5. The rest of the sample continues to migrate to a second binding reagent zone within the control (C) zone, and also forms a second distinct red line (positive control). Regardless of whether any E.coli O157 is present or not, this distinct red line is always formed in the control (C) zone, thus ensuring the test is working correctly.
Storage/Stability
Singlepath®E.coli O157 is stable until the expiry date printed on the box, when stored at +2 to +8°C.
Materials required for the test
Package contents
25 test devices (individually pouched in aluminium foil)
Additionally required materials and instrumentation
1. Enrichment media, e.g. 1.14582. mEC Novobiocin selective enrichment broth or 1.09205. mTSB Novobiocin selective enrichment broth
2. Stomacher / Stomacher bags
3. Incubators +35°C to +37°C or 41.5°C**
4. Purified water
5. Autoclave
6. Waterbath for boiling of samples
7. Disposable Polypropylene tubes for boiling of samples
8. Disposable plastic transfer pipettes and/or appropriate
micro pipettes and disposable tips for dispensing 1-2 ml (sample for boiling)
and 160 µl (application of boiled sample onto tests)
Sample material / sample enrichment
Mix 25 g solid sample or 25 ml liquid sample with 225
ml enrichment medium* and homogenize with a Stomacher if necessary.
Incubate for 18-24 h at +35°C to +37°C or 41.5°C**.
Allow to cool to room temperature
* For dairy products, mTSB + Novobiocin selective enrichment broth (EMD 1.09205.) is recommended.
For meat and meat products, mEC + Novobiocin selective enrichment broth (EMD 1.14582.) should be used.
** Incubation temperature depends on standard used.
Test procedure
Sample Preparation
1. Transfer approx. 1-2 ml of post-enrichment culture to an appropriate (polypropylene) tube. Cover with a loose-fitting cap.
2. Place tubes in boiling water bath for 15 min.
3. Remove and allow cooling to room temperature ( 18 - 26°C ), prior to use.
Procedure
1. Remove the foil pouches from the required number of Singlepath®E.coli O157 devices. Place the test device(s) on a flat surface and label with appropriate sample identification. (Note: Perform the tests within a period of 2 hours after opening!)
2. Using a micro pipette and disposable pipette tip, draw up 160 µl from the boiled, cooled enrichment. Dispense 160 µl of the sample into the circular sample port on the test device. Alternatively using a disposable transfer pipette, squeeze the pipette bulb, insert the stem into the boiled sample and release pressure on bulb. This will draw sample up into the pipette. Dispense five (5) free falling drops (about 150-160 µl) into the circular sample port on the test device.
3. Observe the test result 20 minutes after applying the sample to the device.
Interpretation of results
The test can be regarded as working correctly if a distinct red line appears in the control zone (C) within 20 minutes.
A sample can be considered POSITIVE if at or prior to 20 minutes, red lines appear on both test (T) and control (C) zones.
A sample can be considered NEGATIVE if no red line appears in the test (T) zone but does appear distinctly in the control (C) zone 20 minutes after application of sample to the device.
Technical specifications
Detection limit
Depending on serogroup, a range of approx. 104-106 bacteria/ml can be regarded as being the lower detection limit.
Interferences
Results obtained to date on numerous food samples indicate that there is no interference of Singlepath®E.coli O157 with food ingredients.
The test has been developed based on using mEC + N and mTSB + N selective enrichment broths from MERCK. Interference from other types of selective enrichment broths and other brands cannot be excluded. In particular use of broth of red-brown color could potentially mask weak signals due to background coloration of the test zone.
