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Material Safety Data Sheet

Organisms

Organisms this product works with:

For this Organism

Other products used in the isolation of Listeria monocytogenes :

Rapid Food Tests

Oxoid Listeria Rapid Test

code: FT0401

Intended Use
The Oxoid Listeria Rapid Test (OLRT) is designed for the detection of Listeria species in food and environmental samples within 48 hours. Food samples include dairy, dessert and confectionery foods, raw vegetables, dry foods and additives, raw and processed food, meats, poultry and fish. Environmental samples include swabs from stainless steel, plastic (polythene, polypropylene or polycarbonate), ceramic, glazed earthen material and glass, rubber, food grade painted surfaces, wood, concrete, cast iron and air filter materials. The test protocol allows results to be available two working days after the sample is received in the testing laboratory. The procedure uses two carefully selected enrichment steps for the maximum recovery and growth of Listeria followed by an immunoassay using a test device. This simple system gives a clear visual result within 20 minutes of the addition of the heated and cooled sample to the test device with no further manipulations being required. OLRT has been validated to ISO 16140 by AFNOR1 and was reviewed by AOAC Research Institute2 which found it to perform according to the specifications mentioned in this insert.

Introduction
Listeria is a genus of Gram-positive, nonsporing bacilli with a DNA G+C content of 36–38%. They have up to 6 peritrichous flagella and are motile when grown at 30°C or below. They are aerobic and facultatively anaerobic, catalase positive and oxidase negative. The genus comprises six species, L. monocytogenes, L. ivanovii, L.innocua, L. welshimeri, L. seeligeri and L. grayi. All Listeria, except L. grayi, variously share 4 flagella antigens A, B, C, and D, of which flagella antigen B is common3. Pathogenic and nonpathogenic Listeria are ubiquitous in nature and can be isolated from soil, vegetables and natural waters as well as from healthy animals and man. They are able to grow over a temperature range of 1–45°C. Consequently, L. monocytogenes is a food poisoning risk to susceptible individuals if present in foods that are subsequently stored at these temperatures for sufficient time for the organism to grow to infectious levels before ingestion. Clinical symptoms include flu-like illness, spontaneous abortion, stillbirth, meningitis, pneumonitis, septicaemia and endocarditis.

L. monocytogenes infections mainly occur in neonates, pregnant women, the elderly and immunocompromised individuals.

Test Principle

  1. Enrichment Broth System
    Culture of the test sample is in two sequential enrichments, taking up to 48 hours. Any Listeria organisms present in the food or environmental sample are selectively enriched using growth conditions which are optimal for flagella expression.
  2. Antigen Extraction
    The second enrichment medium is heated at 80°C for 20 minutes to extract the flagella antigen.Listeria test device
  3. Listeria Device
    The Listeria device contains monoclonal antibodies specific to the B flagella antigen4 that is common to the Listeria spp. indicated earlier. The extracted antigen is added to the pad in the sample window. This contains blue latex labelled with antibody. The extract rehydrates the complex, and the flagella specific antigen reacts, if present, with the antibody. The complex moves through the pad by capillary action to a test strip containing an immobilised line of antibody at position T of the result window. A further reaction between any antigen/latex complex and the fixed antibody results in a blue line at position T of the result window. If no flagella antigen/latex complex is present, no blue line will appear at position T of the result window.
    The Listeria device also provides an internal control feature. The appearance of a blue line in the result window at position C shows the test has been carried out correctly.

Precautions
Because of the possibility of culturing and isolating Listeria monocytogenes, pregnant women are advised not to carry out this test.
Listeria screening and the use of this kit should be restricted to persons with at least a basic knowledge of microbiology and associated hazards.
For laboratory use only.
Standard guidelines for the safe handling and the disposal of infectious micro-organisms should be observed throughout all procedures.
Do not mix kit components from different lots.
Do not use after the expiry date stated on the kit.

Components of the Kit

Oxoid Half Fraser Supplement
FT0401A: 5 vials, each sufficient for 2.25 litres of Oxoid Fraser Broth Broth Base
FT0401M: 50 vials, each sufficient for 225ml of Oxoid Fraser Broth Broth Base

Listeria Devices: 50

Positive Control: 3 vials, nonviable Listeria monocytogenes flagella antigen suspension

Instruction Leaflet

Materials and equipment required but not provided
Oxoid Fraser Broth Base, (CM0895)
Oxoid Buffered Listeria Enrichment Broth (BLEB) Base, (CM0897)
BLEB Selective Enrichment Supplement, (SR0141)
Incubator at 30°C ± 2°C
Water bath at 80°C ± 2°C
Glass test tubes or Bijou bottles of 5–8ml capacity
Sterile water/ethanol 1:1 (v/v).

Storage
The OLRT must be stored at 2–8°C. Under these conditions, the components of the kit will retain activity until the date shown on the box. Do not freeze.

Test Protocols
A) Media Preparation Primary Enrichment – Half Fraser Broth (HFB)

Fraser Broth Base (CM0895) should be prepared in accordance with the manufacturer’s instructions and dispensed into appropriate volumes prior to sterilization. Reconstitute the Half Fraser Supplement in accordance with the manufacturer’s instructions. Aseptically add the appropriate volume of reconstituted Half Fraser Supplement to the dispensed volumes of Fraser Broth Base immediately prior to use.

Secondary Enrichment Broth –Buffered Listeria Enrichment Broth (BLEB)

Buffered Listeria Enrichment Broth (CM0897) should be prepared in accordance with the manufacturer’s instructions and dispensed
into 10ml volumes prior to sterilisation. Reconstitute the BLEB supplement (SR0141E) in accordance with the manufacturer’s instructions. Add 40µl of the reconstituted BLEB supplement to 10ml of BLEB base immediately prior to use.

B) Sample Culture System
Food samples should be collected according to local standard methods and guidelines. Prepare by making a homogenate of the food in the HFB, as described in Table 1. (Food samples larger than 25g were not tested in the AFNOR validation).

Environmental swabs should be quenched in 10ml of an appropriate neutralizing agent and the whole added to 90ml of HFB (Table 1).

  1. Dilute the test sample 1 in 10 weight/volume of the prepared Half Fraser Broth and homogenize for the appropriate time according to the  sample type.
  2. Incubate according to the standard (AOAC validated) method or AFNOR validated method as shown below:

    Standard (AOAC validated) method
  3. Incubate the mixture at 30°C ± 2°C for 21–26 hours.
  4. Transfer 0.1ml of the incubated HFB into 10ml of prepared BLEB.
  5. Incubate the inoculated BLEB at 30°C ± 2°C for a further 21–26 hours.

    AFNOR validated method
  6. Incubate the mixture at 30°C ± 2°C for 24–26 hours
  7. Transfer 0.1ml of the incubated HFB into 10ml of prepared BLEB.
  8. Incubate the inoculated BLEB at 30°C ± 2°C for a further 24–26 hours.

C) Preparation of the BLEB sample for testing

  1. Carefully remove the incubated BLEB from the incubator without disturbing any food debris present. Transfer 2ml of the upper region of the broth to a small glass tube.
  2. Place the glass tube in the water bath at 80°C ± 2°C for 20 minutes.
  3. Allow the extract to cool to room temperature.

D) Listeria Device Test Protocol

  1. Ensure that the device is at room temperature before beginning the assay.
  2. Remove a test unit from the foil wrapper and place on a level surface. Label with the identity of the test sample.
  3. Pipette 135µl of the BLEB extract onto the sample window. If any precipitate or viscous material is present after heating, avoid pipetting this into the device.
  4. After 20 minutes, examine for the presence of a blue line of any intensity at position T of the result window.

Interpretation of the Results
A blue line at position T of the result window indicates the presence of Listeria flagella antigen in the BLEB. This indicates a presumptive positive sample.

Confirmation
Subculture from the non heated incubated BLEB broth onto a selective agar, such as Oxford, PALCAM, OCLA (ISO) - formulation according to Ottaviani and Agosti or Brilliance™ Listeria Agar. Incubate the plates according to the manufacturer’s instructions. Results can be confirmed according to AFNOR by following EN ISO11290-1 or by using Brilliance Listeria Agar as follows:

AFNOR EN ISO 11290-1 method of confirmation: Purify suspect colonies on Tryptone Soya Yeast Extract Agar and follow the EN ISO 11290-1 method for confirmation.

AFNOR Brilliance Listeria Agar method of confirmation: Typical colonies (blue/green colonies with or without halo) can be considered as confirmed Listeria species. On Brilliance Listeria Agar, L. monocytogenes grow as blue colonies with an opaque white/cream halo, other Listeria spp. grow as blue colonies without a halo. In the case of discrepant results (positive by OLRT but not confirmed by culture), sufficient steps should be taken to ensure correct and valid results are reported. If the AFNOR validated procedure is not being followed for confirmation, or isolates need to be identified to species level, colonies resembling Listeria should be purified on non selective media and appropriate confirmation and speciation tests carried out.

Device Control
A blue line at the position C of the result window indicates that the device has worked correctly. Differences in the intensity of the blue lines at the positions T and C of the result window may occur, but this does not affect the interpretation of the result. A very strong blue line at the position T of the result window with no line at position C indicates an excess of flagella antigen. This is an uncommon event (typically less than 0.1%). If reassurance that the test has worked properly is required, the sample may be retested with a dilution step. The heated extract should be diluted 1 in 10, in fresh BLEB, and retested on another device. If no line appears at the position T or C of the result window within 20 minutes, a further Listeria device should be tested using the same culture extract, providing this is not more than 1 hour old.

Positive and Negative Controls
A positive (nonviable) flagella antigen freeze-dried control is included in the kit. Reconstitute the vial with 2ml of sterile distilled water. The reconstituted positive control should be stored at 2–8°C.Under these conditions, it will retain its activity for 6 months. For a positive control test, add 135µl of positive control reagent onto the sample window of a separate device and check that a blue line appears at both the positions T and C of the result window within 20 minutes. If a negative control is required, add 135µl of uninoculated BLEB onto the sample window of a separate device and check that a blue line appears only at position C (no blue line should appear at position T). Do not use any other reagents as a negative control.

Limitations of the Oxoid Listeria Rapid Test
The OLRT device is only recommended for use with the Oxoid enrichment broths and supplements that are described in the above protocol5.
Negative results may occur if the amount of antigen extracted is below the minimum sensitivity of the test. The ideal incubation temperature for the expression of Listeria antigens is 30°C. Higher temperatures may affect the overall sensitivity of the method. False-negative results may be obtained when testing concentrated yeast materials by this method. In this case, the incubation time for the Standard method should be a minimum of 24 hours for both primary and secondary enrichment broths. Independent evaluations conducted by AOAC Research Institute were found to verify the claims of the method for the detection of Listeria spp. from all foods with the exception of raw beef and animal feed. See the Validation section for further details. Listeria grayi is not detected.

Performance Characteristics
OLRT has been evaluated in a wide variety of foods, comparing the method with isolation of Listeria spp. on Oxoid Listeria Selective Agar (Oxford Formulation) from the secondary enrichment broth. More than 1000 tests have shown a correlation of >99%6.

Results of an independent evaluation of 62 artificially contaminated samples comprised of a wide variety of surface types demonstrated a sensitivity rate of approximately 80%. Even though the sensitivity rate for Listeria is lower for surfaces, it is comparable to results obtained using the US FDA Bacteriological Analytical Manual method.

AFNOR studies (data on file at Oxoid) show a sensitivity and specificity of 97% and 100%, respectively.
The antibodies in the device have been tested for cross-reactivity against a panel of organisms listed below. The organisms were at a minimum level of 1 x 108/ml. No cross-reactivity was observed with any of the organisms:

Organism tested

Klebsiella pneumoniaeAerococcus viridans
Acinetobacter anitratusBacillus licheniformis
Pseudomonas aeruginosaBacillus circulans
Escherichia coliStaphylococcus aureus
Bacillus cereusStreptococcus faecalis
Salmonella spp.Bacillus subtilis
Enterobacter aerogenesBacillus macerans
Citrobacter koseriEdwardsiella tarda
Enterobacter cloacaeHafnia alvei
Enterococcus faecalisProteus stuartii

Non-Listeria organisms – 7 aesculin positive isolates from Oxford agar.

Limit of Detection
Independent studies (AFNOR, preliminary study, 2007 (ISO 16140)and EMMAS Assessment, 1998) have shown that L. monocytogenes and L. innocua were consistently detected at levels of <10 cells/25grams of food from a variety of food samples, including raw milk,cheese, cured meat, smoked fish and raw vegetables. Further studies showed L. monocytogenes was consistently detected at the lowest challenge levels of 2–7 cells/gram of food. Listeria innocua was detected at the lowest level of 10 cells/gram of food and L. seeligeri was detected at levels of 3cells/gram of food. These levels meet the minimum detectable limits as defined by AOAC Research Institute. This data was verified by an independent testing laboratory.

Validation
OLRT has the following certifications for detection of Listeria spp., AFNOR (Certificate number: UNI-03/02-04/95) according to ENISO 16140 against reference method EN ISO 11290-1/A1 for all food and environmental samples. AOAC RI (Certificate number: 960701) for all food (with the exception of raw beef and animal feed) and environmental samples. A limited selection of foods were independently evaluated by AOAC RI and found to verify the AFNOR results for all foods with the exception of raw beef and animal feed. A limited study and a second, fuller remedial study demonstrated a recovery of around 55% in raw beef samples spiked with low concentrations of Listeria, which is too low for certification. Recovery rates of Listeria spp. from animal feed samples were also found to be unacceptably low at around 67%.

1. AFNOR Certificate No. UNI-03/02-04/95
2. AOAC-RI Certificate No. 960701
3. Seeliger, H. P. R., Jones, D. Genus Listeria Pirie. In: Sneath, P. H. A., Mair, H. S., Sharp, N. E., Holt, J. G. (Eds): Bergey’s Manual of Systematic Bacteriology. Williams & Wilkin Co. Baltimore, 1986.
4. Parry, S. H., Briggs, T., Blades, J. A., Garni, M. and Piron, J. (1993). A rapid Clearview Immunoassay for detection of Listeria. 7th International Congress on Rapid Methods and Automation in Microbiology and Immunology.
5. Holbrook, R., Briggs, T. A., Anderson, J. A., Blades, J. A. and Sheard, P. N. (1993). Detection of Listeria species in foods in 43 hours using enrichment and the Listeria Clearview™ Immunoassay. 7th International Congress on Rapid Methods and Automation in Microbiology and Immunology.
6. Holbrook, R., Briggs, T., Anderson, J., Blades, J. and Sheard, P. N. (1994). A 43 hour Test for Detecting Listeria in Foods using the Unipath Clearview Immunoassay. 81st Annual Meeting of the International

 
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