TECRA Listeria Visual Immunoassay - AOAC 2002.09

TECRA Listeria Visual Immunoassay - AOAC 2002.09

SCOPE

This method is applicable to detection of Listeria spp. in raw and processed meats. The method may also be used for detection of Listeria spp. in environmental samples. The method varies from AOAC 995.22 in the enrichment broth used.

PRINCIPLES

Detection of Listeria antigens is based on an enzyme-linked immunosorbent assay (ELISA) performed in a “sandwich” configuration. Highly specific antibodies adsorbed to wells capture Listeria antigens in an enriched sample. Captured antigens are bound to a conjugate which is exposed to a substrate that results in a green colour being developed when suspect antigens are present. Determination of presumptive positive results can be performed either visually or spectrophotometrically.

Primary enrichment

Raw meat samples are diluted 1:10 in TLEB [1] and incubated at 35-37C for 24 h. Processed meat samples are diluted 1:20 in TLEB base and incubated at 35-37C for 4 h before adding TECRA Listeria selective supplement. Samples are then incubated for a further 20 h at 35-37C. Environmental samples can be enriched in 60-100 ml TLEB for sponges or in 10 ml if using small swabs.

Secondary enrichment

Primary enrichments from raw meat samples are transferred into Fraser broth and incubated at 30C for 24 h. Primary enrichments form processed meat samples are transferred into fresh TLEB (complete medium) and incubated at 30C for 24 h.

Immunosorbent assay

Listeria is detected in boiled test broths using TECRA Listeria Visual Immunoassay (following the manufacturer’s instructions).

Cultural confirmation

All Listeria positive samples must be confirmed using AS 5013.24.1. Confirmation must be carried out using secondary enrichment broth at the appropriate stage in AS 5013.24.1 ie plating out and identification.

Issue 2015 10 27 | Approved Methods Manual

Export Standards Branch | Exports DivisionPage 1 of 2

Department of Agriculture and Water Resources

Listeria Detection - TECRA – AOAC 2002.09

CHECKLIST

Primary-Enrichment / Is primary enrichment carried out in TLEB?
Are the appropriate enrichment protocols used for the type of product sampled?
Are samples enriched at 35-37C for 24 h?
What volume of TLEB is added to environmental swabs?
Is a positive control run with each batch of samples analysed?
Are reference cultures inoculated into primary enrichment broth at a level of 10 to 100 cells?
Secondary-Enrichment / Are primary enrichments transferred into Fraser broth or fresh TLEB for raw and processed meats respectively?
Is secondary-enrichment carried out at 28-30C for 22-24 h?
Are secondary enrichment broths stored at 2-8C for possible confirmation of positive results?
Immunoassay / Is the TECRA assay performed on boiled broth (boiling water for 10-15 min) cooled to 25-37C?
Are the manufacturer’s instructions available and are they reproduced in the laboratory manual?
Is initial incubation of kits carried out at 35-37C?
Are kit components stored at 2-8C when not in use?
Are kits warmed to room temperature before use?
Confirmation / Is Listeria confirmed using AS 5013.24.1, MLG 8.08 or FDA BAM Chapter 10?
(if applicable) / If an external laboratory is used is it department approved?

Issue 2015 10 27 | Approved Methods Manual

Export Standards Branch | Exports DivisionPage 1 of 2

Department of Agriculture and Water Resources

[1] TLEB (TECRA Listeria Enrichment Broth) is a proprietary media for use with TECRA Visual Immunoassay kits. It does not contain the highly toxic antifungal agent, cycloheximide and is available as a complete medium or a base medium to which selective supplements are added.