Duopath® Verotoxins

EMD Cat. No. 1.04144.0001
25 tests


GLISA-Rapid Test (Gold Labelled ImmunoSorbent Assay) for the qualitative detection of verotoxins from verotoxin-forming E.coli.

Introduction
 Amongst the E.coli human pathogens, verotoxin (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, O26, O10³, O111, O145, and other strains are particularly interesting in this respect. Production of verotoxins is the most common criteria for the detection of this group of bacteria. Verotoxins can be classified in the main category verotoxin 1 (VT1, SLT1, Stx1) and 2 (VT2, SLT2, Stx2). EHEC strains are able to produce either VT1 or VT2 only or even both VT1 and VT2 simultaneously.
 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.
 Duopath® Verotoxin VT1/VT2 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.

Principle
 Duopath® Verotoxins ( 1.04144) is an immunochromatographic rapid test based on gold-labelled antibodies. The test device has a circular sample port, and an oval shaped test (VT1, VT2) 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 verotoxins.
 3. Any verotoxin (VT1 and VT2) antigen present complexes with the gold-labelled antibody  and migrates through the port until it encounters the binding zones in the test (VT1, VT2) area.
 4. The binding zones (VT1 and VT2) contain  another anti- VT1 or VT2 Antibody, which immobilises any verotoxin -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 another binding reagent zone within the control (C) zone, and also forms a further distinct red line (positive control). Regardless of whether any verotoxin 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
 Duopath® Verotoxins 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 Selective Enrichment Broth w/ Novobiocin or 1.09205. mTSB Selective Enrichment Broth w/ Novobiocin, 1.09207. SMAC Agar, 1.09202.CT-Supplement, 1.00060. CAYE Broth mod. acc.to Evans, 1.00051. CAYE Broth Supplement
 2. Polymyxin - Solution: 5 mg Polymyxin-B-sulfate in 1 ml of deionized water
 3. Stomacher/Stomacher bags
 4. Incubators +35°C and +41.5°C
 5. Distilled or deionized water
 6. Autoclave
 7. Disposable plastic transfer pipettes and/or appropriate micro pipettes and disposable tips for dispensing 160 µl

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 +41.5°C.
 Inoculate CT-SMAC Agar** with an aliquot from the enrichment broth.
 Incubate for 18-24 h at +35 to +37°C.
 Pick up 5-6 typical colonies from the CT-SMAC Agar and dispense it in 1 ml of the CAYE broth containing CAYE broth supplement.
 Incubate for 6 h at +37°C.
 Pipette 180 µl CAYE-culture into a Eppendorf Cup.
 Add 20 µl Polymyxin-solution and mix.
 Incubate the Eppendorf Cup for 10 min. at 35-37°C.
 Allow to cool to room temperature.
 * For dairy products, mTSB + Novobiocin selective enrichment broth (MERCK 1.09205.) is recommended.
 For meat and meat products, mEC + Novobiocin selective enrichment broth (MERCK 1.14582.) should be used.
 **Only  E.coli O157 serovars are able to grow on CT-SMAC Agar. To detect verotoxins of other serovars other differential media must be used, e.g. SMAC Agar.

Test procedure
 Sample Preparation
 Prior to use, allow enriched sample and test devices to reach room temperature (+15 to +25°C).
 Procedure
 1. Remove the foil pouches from the required number of Duopath® Verotoxins 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. Briefly stir the Eppendorf Cup with a Vortex mixer.
 3. Using a micro pipette and disposable pipette tip, draw up 160 µl and it into the circular sample port on the test device.
 Alternatively using a disposable transfer pipette, squeeze the pipette bulb, insert the stem into the Eppendorf Cup 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.
 4. 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 15-20 minutes, red lines appear on both test (VT1 and/or VT2) and control (C) zones.
 A sample can be considered NEGATIVE if no red line appears in the test (VT1 and VT2) zone but does appear distinctly in the control (C) zone 15-20 minutes after application of sample to the device.

Technical specifications
 Detection limit
 One colony 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 Duopath® Verotoxins with food ingredients.
 The test has been developed based on using CAYE medium from MERCK. Interference from other types of CAYE medium 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.

Trouble-shooting
Problem
 		Measures
No line appears in either zone after 20 minutes test period.
Re-run sample.



Duopath® Verotoxins



© 2002 Merck KGaA, Darmstadt, Germany