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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