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Toxin Detection Kits

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VTEC-RPLA TOXIN DETECTION KIT

Code: TD0960

A reverse passive latex agglutination test for the detection of verocytotoxins VT1 and VT2 produced by Escherichia from cultured from food and faecal samples.

 INTRODUCTION
Verocytotoxin producing E. coli (VTEC) are transmitted through food, water and person - to-person contact, and are known to cause a range of illnesses from self limiting watery diarrhoea and haemorrhagic colitis to haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura.1,2,3,4,5
These illnesses can be fatal, making the increasing incidence of VTEC contamination a cause for widespread concern.
Unlike other tests which detect the presence of strains such as E. coli 0157 (of which some, but not all, produce verocytotoxins), the Oxoid VTEC-RPLA test detects the toxins themselves, providing a clear and specific indication of VT1 or VT2 production. This overcomes the problem of positive results from other latex and culture assays (i.e. those which detect the organism rather than the toxin) where non-toxin-producing E. coli 0157 strains are present. Similarly, it also overcomes the problem of negative results in cases where non-0157 strains are responsible for toxin production.
The test can be used with isolates cultured from both food and faecal samples.

PRINCIPLE OF THE ASSAY
Polymer latex particles are sensitised with purified rabbit anti-serum which is reactive either with E. coli verocytotoxin VT1 or VT2. The latex particles will agglutinate in the presence of one or both of the E. coli verocytotoxins. Agglutination results in the formation of a lattice structure. On settling, this forms a diffuse layer on the base of a V-bottom microtitre well. If E. coli verocytotoxin is absent, or at a concentration below the assay detection level, no such lattice structure can be formed, and a tight button will therefore be observed. The use of polymyxin B solution facilitates the release of verocytotoxins.6,7

SAMPLE PREPARATION
E. coli isolates are recovered and identified from food or clinical samples using standard procedures.
Serogroup 0157 organisms may be identified using Sorbitol MacConkey Agar (Oxoid CM813), since they are unable to ferment sorbitol. Other serogroups of E. coli do, however, produce verocytotoxin.

 METHOD OF USE
1. Materials required but not provided:
Microtitre plates (V-well) and lids.
Fixed or variable pipette and tips 25m l.
Centrifuge capable of generating 900 g (typically 4,000 rpm in a small bench-top centrifuge).
Membrane filtration unit using low protein-binding disposable filters with a porosity of 0.2 m m - 0.45 m m (such as Millipore SLGV).
CA-YE Broth or Brain Heart Infusion Agar (Oxoid CM375).
Polymyxin B (see 3.2.2).
Sodium chloride solution (0.85%).
Sodium hypochlorite solution (1.3% w/w).
25m I dropper (optional).
25m 1 diluter (optional).
Micromixer (optional).
Moisture box (optional).

2. Components of the kit:
TD 961
Sensitised latex VT1 Latex suspension sensitised with specific antibodies (rabbit IgG) against E. coli verocytotoxin type 1.
TD 962 Sensitised latex VT2 Latex suspension sensitised with specific antibodies (rabbit IgG) against E. coli verocytotoxin type 2.
TD 963 Latex Control Latex suspension sensitised with non-immune rabbit globulins.
TD 964 Verotoxin Control (VT1) Dried E. coli verocytotoxin type 1.
TD 965 Verotoxin Control (VT2) Dried E. coli verocytotoxin type 2.
TD 966 Diluent Phosphate buffered saline.
Instruction leaflet

3.Toxin Production and Extraction
E. coli may be tested for verocytotoxin production by growth in culture media.Growth of the organism may be performed in a liquid medium (CA-YE broth), or on a Brain Heart Infusion Agar. Growth on Agar is followed by extraction in a polymyxin B solution.

3.1 Broth culture method
3.1.1
Isolated organism is inoculated into CA-YE broth and incubated at 37° C for 18-20 hours with vigorous shaking (120-150 oscillations per minute).
3.1.2 After growth, the culture is either centrifuged at 4,000 rpm for 20 minutes at 4° C or filtered using a 0.2m m -0.45m m low protein binding filter (such as Millipore SLGV). The filtrate is retained for the verocytotoxin assay.

3.2 Solid culture method
3.2.1
Isolated organism is inoculated onto Brain Heart Infusion Agar (Oxoid CM375) slopes (10ml volumes) and incubated at 37° C for 18-20 hours.
3.2.2 After incubation, the growth is removed using a microbiological loop and suspended in 1 ml of a 0.85% sodium chloride solution containing polymyxin B at a concentration of 5,000 units per ml.
3.2.3 Extraction is continued for 30 minutes at 37° C, shaking occasionally.
3.2.4 After extraction the culture is either centrifuged at 4,000 rpm for 20 minutes at 4° C or filtered using a 0.2m m - 0.45m m low protein binding filter.The filtrate is retained for the verocytotoxin assay.

4. Control
Each reconstituted toxin control will cause agglutination with its homologous test latex. The use of the toxin controls will provide reference for the positive patterns illustrated below (see INTERPRETATION OF TEST RESULTS). The controls need only be used from time to time in order to confirm that the test latex reagents are working correctly. The toxin control is not provided at a specified level and must not therefore be used as a means of quantifying the level of toxin detected in the test sample.

5. Assay method
5.1
The latex reagents and diluent are ready for use. The latex reagents should be thoroughly shaken before use to ensure an homogeneous suspension. To reconstitute the control toxins, 0.5ml of diluent is added to each vial. The contents are shaken gently until dissolved.
5.2 The plate is arranged so that there are 3 columns, each consisting of 8 wells.
5.3 Using a pipette or dropper, 25m l of diluent is dispensed into each well.
5.4 25m l of test sample supernatant is added to the first well of each column.
5.5 Starting at the first well of each column, a pipette or diluter is used to pick up 25m l and perform doubling dilutions down each column, up to and including row 7. (NB: 25m l of sample and Buffer mix must be removed from the 7th well and discarded). The last row of wells is left containing diluent only.
5.6 25m l of the test latex VT1 is added to each well in the first column.
5.7 25m l of the test latex VT2 is added to each well in the second column.
5.8 25m l of latex control is added to each well in the third column for the purpose of detecting false agglutination reactions.
5.9 Toxin controls are assayed in the same manner as test samples.
5.10 The contents of each well are mixed either by rotating the plate using a micromixer or agitating by hand. Care must be taken to avoid spillage.
5.11 To avoid evaporation, the plate is covered with a lid. (Alternatively, it may be placed in a moisture box). The plate is left undisturbed on a vibration-free surface at room temperature for 20-24 hours. To assist subsequent reading of the test, the plate may be placed on black paper for the incubation period.
5.12 Each well in each column is examined for agglutination against a black background.
5.13 Centrifuge tubes, membrane filters, microtitre plates, lids and pipette tips should be sterilised by autoclaving at 121° C or disinfected before disposal in hypochlorite solutions (>1.3% w/w).
5.14 Culture extracts, food extracts, samples and toxin controls should be disposed of in hypochlorite solutions (>1.3% w/w).

INTERPRETATION OF TEST RESULTS
The agglutination pattern should be judged by comparison with the following illustration:

Results classified as (+), (++) and (+++) are considered to be positive. Results classified as () and (-) are considered to be negative. Results in the 3rd column of wells containing latex control should be negative as should the 8th row of columns 1 and 2 which contain no sample. If positive patterns are observed in these wells, the test should be regarded as invalid.
In some cases, non-specific agglutination may be observed. In such cases, the results should be interpreted as positive, provided that the reaction with the test latex is positive to a dilution of test sample four times higher than that seen with the latex control.
In samples from high-level verocytotoxin producing strains, negative patterns of agglutination in the lower dilution wells may be observed due to antigen excess (i .e. the prozone effect); since, however, the amount of toxin in each test well is reduced 2-fold at each dilution, inhibition of agglutination is overcome and true positives can be read.

LIMITATIONS OF THE TEST
The sensitivity of the test is 1 to 2 ng/ml of E.coli verocytotoxin. Verocytotoxin present at concentrations lower than this will therefore give negative results. The production of VT1 and VT2 by E. coli isolated from clinical or food sample does not constitute a diagnosis of disease. Strains producing verocytotoxin have been isolated from healthy individuals and farm animals. New unused V-well plates should be used, as scratched wells may cause inconsistent results. Reagents with different lot numbers should not be interchanged or mixed.

APPENDIX
CA-YE Broth Formulation8

 

Grams per Litre

Casein hydrolysate (Oxoid L 41 )

20.00

Yeast extract (Oxoid L 21)

6.00

Sodium chloride (NaCI)

2.50

Di-potassium hydrogen phosphate (K2HPO4)

8.71

Salts solution

1 ml

To prepare the salt solution, dissolve the following in distilled water:

Magnesium sulphate (MgSO4.7H20)

5.0%

Manganese chloride (MnCI2.4H20)

0.5%

Ferric chloride (FeCI3.6H20)

0.5%

Adjust the pH to 8.0 - 8.2 with 0.1N NaOH, dispense into suitable volumes (2-10 ml), then autoclave at 121°C for 15 minutes.

REFERENCES
1.
Centers for Disease Control -United States, (1982) Morb. Mortal. Wkly. Rep., 31:580-585.
2. Konowalchuk, J., Speirs, J.I. and Stavric, S.(1977) Inf. Immun., 18:775-779
3. Karmali, M.A., Steele, B.T., Petric, M. and Lim, C (1983) Lancet 1:619-620
4. Pal, C.H., Gordon., Sims, H.V. & Briant, L.E., (1984) Ann. Intern. Med., 101:738-742
5. Waters, J.R., (1985) Con. Dis. Wkly.Rep., 11:123-124
6. Karmali, M.A., Petric, M., Lim C.,Fleming, RC., Arbus, G.S. and Lior, H. (1985) J. InL Dis., 151:775-782
7. Cerny, G. andTenber, M. (1971) Arch.Microbiol. 78:166-179
8. Evans, D.G., Evans, D.J. and Gorbach, S.L. (1973) Inf. and Immun., 8: 731-735

 
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