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Toxin Detection Kits

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BCET-RPLA TOXIN DETECTION KIT

Code: TD0950

A kit for the detection of Bacillus cereus enterotoxin (diarrhoeal type) in foods and culture filtrates by reversed passive latex agglutination.

INTRODUCTION
In both its spore and vegetative forms, the organism Bacillus cereus is a common inhabitant of many different environments and can easily contaminate food. If contaminated foods are not cooled sufficiently after cooking and there is an extended time between preparation and consumption of foods, then surviving heat-resistant spores can germinate, enabling the organism to multiply and produce toxins. Under these circumstances, B cereus can cause food poisoning. Rice, pasta, meat, poultry, vegetable dishes, various soups, puddings and sauces have been implicated in B. cereus food poisoning.1,2,3.
Two distinct types of illness can be caused by this organism: the acute-onset ‘emetic-syndrome’ type which is mainly associated with cooked rice; and the longer-onset ‘diarrhoeal-syndrome’ type in which a wide range of foods have been implicated. Separate toxins are responsible for the characteristic symptoms of the two forms of illness: the emetic toxin and the diarrhoeal enterotoxin4.
This kit was developed for the purpose of detecting the diarrhoeal enterotoxin by reversed passive latex agglutination (RPLA). The technique of reversed passive latex agglutination (RPLA) enables soluble antigen such as bacterial toxins to be detected in an agglutination assay.
In a standard agglutination assay, soluble antibody reacts with particulate antigen such as bacterial cells. However, in a REVERSED agglutination assay the antibody, which is attached to particles, reacts with the soluble antigen. The particles (in this case, latex) do not themselves play a part in the reaction and they are therefore PASSIVE. The cross-linking of the latex particles by the specific antigen/antibody reaction results in the visible LATEX AGGLUTINATION reaction.
The BCET-RPLA test may be used to detect B. cereus enterotoxin in a variety of foods and to give a semi-quantitative result. The test may also be used to demonstrate enterotoxin production by isolates of B. cereus grown in culture.

PRINCIPLE OF ASSAY
Polystyrene latex particles are sensitised with purified antiserum taken from rabbits immunised with purified B. cereus diarrhoeal enterotoxin. These latex particles will agglutinate in the presence of B. cereus enterotoxin. A control reagent is provided which consists of latex particles sensitised with non-immune rabbit globulins. The test is performed in V-well microtitre plates. Dilutions of the food extract or culture filtrate are made in two rows of wells, a volume of the appropriate latex suspension is added to each well and the contents mixed. If B. cereus enterotoxin is present, agglutination occurs due to the formation of a lattice structure. Upon settling, this forms a diffuse layer on the base of the well. If B. cereus enterotoxin 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.

PRECAUTIONS
This product is for in vitro diagnostic use only.
Do not freeze.
Reagents with different lot numbers should not be interchanged.
Reagents and diluent contain 0.1% sodium azide as a preservative. Sodium azide may react with lead or copper plumbing to produce metal azides which are explosive by contact detonation. To prevent azide accumulation in plumbing, flush with copious amounts of water immediately after waste disposal.

STORAGE
The BCET-PRLA kit must be stored at 2-8° C. Under these conditions the reagents will retain their reactivity until the date shown on the kit box. After reconstitution, the enterotoxin control should be stored at 2-8° C. Under these conditions, the reconstituted enterotoxin control will retain its reactivity for 3 months, or until the date shown on the kit box, whichever is the sooner.

SAMPLE PREPARATION
Food Matrices
A wide range of foods may be tested for enterotoxin. The extraction procedure may, however, require modifications for particular foods. The main requirement is to achieve a non-turbid, fat-free extract. A low dilution factor is desirable for optimum sensitivity, but if the nature of the food dictates a greater dilution during extraction, a reduced sensitivity will result.
To gain a representative sample of a batch, a series of 10g portions are collected from different locations within the batch (see T.P.I., U.S.D.A. sampling plans or equivalent).
Culture Filtrates
B. cereus may be recovered from food or faecal samples and identified using suitable techniques described in standard textbooks. The use of Bacillus Cereus Selective Agar (Oxoid CM617 and SR99) will aid the isolation and presumptive identification of B. cereus prior to toxin detection5.

METHOD OF USE
Materials required but not provided.
Blender or homogeniser (required for food matrices only).
Microtitre plates (V-well) and lids
Fixed or variable pipette and tips (25µg)
Centrifuge capable of generating 900g (typically 3000rpm in a small bench top centrifuge) or membrane filtration unit using low protein-binding disposable filters with a porosity of 0.2µm to 0.45µm (such as Millipore SLGV)
Brain Heart Infusion (Oxoid CM225)
Sodium chloride solution (0.85%)
Sodium hypochlorite solution (>1.3% w/w( (disinfectant)
25µl dropper (optional)
25µl diluter (optional)
Moisture box (optional)

Components of the Kit
TD951 Sensitised Latex.
Latex sensitised with specific B. cereus anti-enterotoxin (rabbit lgG).
TD952 Latex control. Latex suspension sensitised with non-immune rabbit globulins.
TD953 Enterotoxin control (lyophilized). Lyophilized B. cereus enterotoxin.
TD954 Diluent. Phosphate buffered saline containing bovine serum albumin.
Instruction leaflet

Toxin Extraction or Production
Extraction from Food Matrices
Blend 10g of sample with 10ml of sodium chloride solution (0.85%) in a blender or homogeniser.
Centrifuge the blended sample at 900g at 4°C for 30 minutes. NOTE: If refrigerated centrifuge is not available, cool the sample to 4°C before centrifugation.
Filter the supernatant through a 0.2µm-0.45µm low protein-binding membrane filter. Retain the filtrate for assay of toxin content.
Production of Enterotoxin in Culture Fluids
Inoculate the isolated organism into Brain Heart Infusion (CM225) and incubate at 32-37° C for 6-18 hours, preferably with shaking (250 cycles/min).
After growth, either centrifuge at 900g for 20 minutes at 4°C or membrane filter using a 0.2µm-0.45µm low protein-binding filter.
Retain the filtrate for assay of toxin content. NOTE: It is advisable to check the particular cultural method of use with a standard enterotoxin-producing strain such as B. cereus NCTC 11145.

Control
The reconstituted toxin control will agglutinate the sensitised latex. The use of the toxin control will provide a reference for the positive patterns illustrated below (see interpretation of Test Results). The control should be used from time to time only to confirm the correct working of the test latex. The toxin control is not provided at a specified level and therefore must not be used as a means of quantifying the level of toxin detected in the test sample.

Assay Method
Working Reagents
The latex reagents (TD951, TD952) and diluent (TD954) are ready for use. The latex reagents should be thoroughly shaken before use to ensure a homogenous suspension. To reconstitute the enterotoxin control (TD953), add 0.5ml of diluent (TD954) to each vial. Shake gently until the contents are dissolved.
Arrange the plate so that each row consists of 8 wells. Each sample needs the use of 2 such rows.
Using a pipette or dropper dispense 25µl of diluent in each well of the 2 rows except the first well in each row.
Add 25µl of test sample to the first and second well of both rows.
Using a pipette or diluter and starting at the second well of each row, pick up 25µl and perform doubling dilutions along each of the rows. Stop at the 7th well to leave the last well containing diluent (TD954 only).
To each well in the first row add 25µl of sensitised latex (TD951).
To each well in the second row add 25µl of latex control (TD952).
To mix the contents of each well, rotate the plate by micromixer or agitate by hand. Take care that no spillage occurs from the wells.
To avoid evaporation, cover the plate with a lid. Placing the plate in a moisture box is an acceptable alternative. Leave the plate undisturbed on a vibration-free surface at room temperature for 20 to 24 hours. It will help subsequent reading of the test if the plate is placed on black paper for the duration of this incubation.
Examine each well in each row for agglutination against a black background.
Centrifuge tubes, membrane filters, microtitre plates, lids and pipette tips should be sterilised by autoclaving at 121°C for 15 minutes or disinfected, before disposal, in hypochlorite solutions(>1.3%w/w).
Dispose of culture extracts, food extracts, samples and enterotoxin controls in hypochlorite solution (>1.3%w/w).

INTERPRETATION OF TEST RESULTS

The agglutination pattern should be judged by comparison with the following illustration:

Results classified as (+), (++) are considered to be positive.
Results in the row of wells containing latex control (TD952) should be negative. In some cases, non-specific agglutination may be observed. In such cases the results should be interpreted as positive, provided that the reaction with sensitised latex (TD951) is positive to a higher dilution of test sample that that seen with the latex control (TD952). The last well in all rows should be negative. If positive patterns are observed in some of these wells, the reaction should be regarded as invalid.

LIMITATIONS OF THE TEST
The sensitivity of this test in detecting the enterotoxin is 2ng/ml in the test extract. When a food extract is made with a dilution ratio of 1:1 with diluent (TD954), the sensitivity is, therefore, 4ng/g of food matrix. The detection limit will vary according to any extra dilution conditions dictated by the type of food matrix. Concentration of the enterotoxin in the food extract can be effected by a variety of methods, such as ultrafiltration.
Production of enterotoxin in culture filtrate depends on the growth conditions. A positive result obtained in this way demonstrates the production of enterotoxin; it does not imply the in vitro production of toxins to those levels.

REFERENCES
1.
Kramer, J.M. and Gilbert; R.J. (1988). In Foodborne Bacterial Pathogens (ed. M.P. Doyle) pp. 21-70 Marcel Dekker Inc,. New York
2. Hauge, S. (1955) J. Appl. Bacterial 18: pp. 591-595.
3. Mortimer, P.R. and McGann, G. (1974). Lancet 1: pp. 1043-1045
4. Turnbull, P.C.B. (1936). In Pharmacology of Bacterial Toxins (ed. F. Dorner and J. Drews) pp. 397-448. Pergamon Press, Oxford.
5. Holbrook, R. and Anderson, J.M. (1980). Can. J. Microbiol. 26: pp. 753-759.

 
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