E. coliPRO™ O157 KIT

Cat. no. PL070HD E. coliPRO™ O157 Kit 50 tests/kit
Each kit contains:
E. coliPRO™ O157 Latex Reagent 1 dropper vial/kit
E. coliPRO™ O157 Positive Control Suspension 1 dropper vial/kit
E. coliPRO™ O157 Negative Control Latex Reagent 1 dropper vial/kit
Latex Test Cards 20 cards/kit
Mixing Sticks 75 sticks/kit

INTENDED USE

The Hardy Diagnostics E. coliPRO™ O157 Kit provides a rapid latex agglutination method for the detection of E. coli serogroup O157 antigen from colonies isolated from laboratory culture media.

SUMMARY

Escherichia coli serotype O157:H7 is a verotoxin-producing (VT-producing) pathogen. This serotype has been reported as an etiological agent in sporadic and outbreak cases of hemorrhagic colitis. It is also associated with hemolytic uremic syndrome. Certain E. coli serotypes, other than O157:H7, also produce verotoxin. However, the diarrhea caused by these other serotypes is not usually bloody. Additionally, E. coli serotype O157:H7 does not ferment sorbitol. Therefore, if MacConkey Agar with Sorbitol is used as a primary screen, the colonies of E. coli serotype O157:H7 appear colorless as they are non-sorbitol-fermenting, while colonies of other serotypes are pink indicating sorbitol-fermentation.(1-4)

Hardy Diagnostics E. coliPRO™ O157 Kit contains blue latex particles coated with an antiserum against E. coli O157 antigen. When the coated latex particles are mixed with fresh colonies of E. coli serotype O157 the bacteria will bind to the antiserum, causing the latex particles to visibly agglutinate, indicative of a positive reaction. Bacteria which do not belong to the O157 serotype will not bind to the antiserum and will not result in agglutination, indicative of a negative reaction.(5,6)

MATERIALS SUPPLIED

E. coliPRO™ O157
Latex Reagent
Blue latex particles coated with purified rabbit IgG which react with E. coli serotype O157, 3.1ml
E. coliPRO™ O157
Positive Control Suspension
E. coli serotype O157:H7 colonies grown on agar medium, harvested and inactivated, 1.5ml
E. coliPRO™ O157
Negative Control Latex Reagent
Blue latex particles coated with purified rabbit IgG which does not react with E. coli serotype O157, 1.5ml
Latex Test Cards Disposable white cards with 10 reaction circles
Mixing Sticks Disposable wooden sticks

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, pasteur pipettes, Saline (Cat. no. K59), timer, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8°C away from direct light. Do not freeze . This kit, or any of its reagents, should not be used if there are any signs of discoloration, contamination, or if the expiration date has passed.

PRECAUTIONS

The Latex Reagents contain sodium azide as a preservative. Sodium azide can react explosively with copper or lead if allowed to accumulate. Although the amount of sodium azide in the reagents is minimal, large quantities of water should be used when flushing these reagents down the sink.

Do not use the Latex Reagents if autoagglutination is visible. Autoagglutination is defined as agglutination of
E. coliPRO™ O157 Latex Reagent in the absence of added test specimen or agglutination of the Negative Control Latex Reagent with the test specimen or Positive Control Suspension. Autoagglutination may indicate that contamination or reagent deterioration has occurred.

PROCEDURE

Specimen Collection:
For specific procedures regarding specimen collection and preparation of primary cultures consult standard microbiological references.(1-4)

In general, the Latex Test should be performed on isolated non-sorbitol-fermenting colonies that are 18 to 24 hours old. Isolates can be taken from MacConkey Agar with Sorbitol (Cat. no. G36) or CT-SMAC Agar plates (Cat. no. G129).

Test Protocol:

1. Allow all reagents to come to room temperature for at least 10 minutes prior to use.

2. Place one drop of sterile Saline (Cat. no. K59) within the circle on the test card.

3. Select 1-4 well-isolated colonies from the agar surface.

4. Create an emulsion of the colonies by mixing the Saline on the test card.

5. Mix the Latex Reagents, by inverting the tubes, prior to use. Dispense 1 drop of E. coliPRO™ O157 Latex Reagent onto a test circle on the test card. Important: Do not allow the Latex Reagent bottle to come into contact with the organism suspension.

6. Mix the Latex Reagent and the organism suspension with the wooden sticks provided, using the complete area of the circle. A new stick should be used for each reagent.

7. Gently hand-rock the entire card, allowing the mixture to flow slowly over the ring area.

8. For up to 2 minutes, under normal lighting conditions, observe for agglutination (strong clumping) of the latex particles.

9. All organisms yielding a positive agglutination reaction should be retested with the Negative Control Latex Reagent. Repeat steps 4-8 with the Negative Control Latex Reagent.

See "Interpretation of Results" and "Limitations" sections for more information.

INTERPRETATION OF RESULTS

Positive Results:

A rapid and significantly strong clumping of the latex particles in under 2 minutes requires additional testing with the Negative Control Reagent to confirm that the observed agglutination reaction was specific for the E. coli O157 serotype.

If the organism agglutinates with the Latex Test Reagent and fails to agglutinate with the Negative Control Latex Reagent, this indicates the identification of E. coli serotype O157.

If the organism agglutinates with the Latex Test Reagent and also agglutinates with the Negative Control Latex Reagent, this is considered a false-positive reaction due to autoagglutination or cross reaction of the strain.

Negative Results:

If no visible agglutination of the latex particles is observed in any of the reaction circles, there is no need to continue testing with the Negative Control Latex Reagent.

LIMITATIONS

The kit is intended for use in the identification of pure cultures from MacConkey Agar based media, which show typical E. coli morphology.

False-negative or false-positive results can occur if inadequate amounts of culture or Latex Test Reagents are used.

Positive test results should be biochemically confirmed. Conventional serological testing with E. coli H antisera should be used to confirm the serotype of a latex agglutination positive culture. Occasionally non-motile E. coli O157 are encountered.

E. coliPRO™ O157 Reagents were developed to detect the presence of E. coli serogroup O157 antigen. Most non-sorbitol-fermenting colonies that give a positive result in this kit are presumptively identified as E. coli O157:H7. Some other E. coli O157 strains, for instance E. coli O157:H16, that are non-sorbitol-fermenting may also produce a positive reaction in this test.

If stringy or mucoid agglutination reactions are observed between the E. coliPRO™ O157 Latex Reagent and the organism suspension, the reactions are considered uninterpretable and retesting of the organism should occur. It is recommended that a fresh organism suspension be prepared and the colony clumps be allowed to settle out of solution. Only the supernatant should be used in retesting.

Although this test has been specifically developed to reduce the normal cross-reactivity of Escherichia hermanii , rare and uncommon strains may cross react. Cellobiose growth in the presence of KCN and yellow pigmentation may be used for differentiation.

QUALITY CONTROL

The following organisms are routinely tested on each lot of the E. coliPRO™ O157 Kit:

Test Organisms
Inoculation Method*
Testing
Time
Results
Escherichia coli O157:H7

ATCC® 700728
*
up to 2 min.
Agglutination observed only with the Latex Reagent
Escherichia coli
ATCC® 25922
*
up to 2 min.
No agglutination observed only with the Latex Reagent

User Quality Control

END USER QUALITY CONTROL

It is recommended that the performance of the Latex Reagent and the Negative Control Latex Reagent be evaluated with the Positive Control Suspension with each new lot number and new shipment. In accordance with the laboratory's quality control program and/or regulatory guidelines, further periodic testing may be required.

The Latex Reagent should show obvious agglutination with the Positive Control Suspension. The Positive Control Suspension contains E. coli O157:H7 antigens from E. coli serotype O157:H7 grown on agar medium, harvested, and inactivated to produce the positive control antigen. If agglutination is observed with the Positive Control Suspension, it is necessary to test the Negative Control Latex Reagent with the Positive Control Suspension to yield no agglutination.

The use of the kit should be continued only if the Latex Reagent agglutinates with the Positive Control Suspension and the Negative Control Latex Reagent fails to agglutinate with the Positive Control Suspension.

If the Latex Reagent fails to agglutinate with the Positive Control Suspension, this indicates the potency of the reagents is low and they need to be discarded.

If the Latex Reagent agglutinates with the Positive Control Suspension and the Negative Control Latex Reagent also agglutinates with the Positive Control Suspension, this indicates the reagents are autoagglutinating and need to be discarded.

REFERENCES

1. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

2. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

5. Broczyk, A., et al. 1987. False-positive identification of Escherichia coli in foods. Int. J. Food Microbiol. 4:347-349.

6. Thompson, J.S., 1990. Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype 0157. J. Clin. Microbiol. 28:2165-2168.


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021716gr

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