1-2 Test The most widely used single unit test for Salmonella AOAC Official Method (989.13)

1-2 Test ® For Salmonella Immunodiffusion (Antibody-based detection method) Requires movement of salmonella specific antibodies and target antigen through semi-solid gel medium Antibody movement - diffusion Target antigen movement – motility of live Salmonella cells The 1-2 Test is the most widely used test for Salmonella screening in the world. This patented single-use unit is completeyl self-contained. The 1-2 test uses a unique combination of a built-in selective enrichment, immunodiffusion and a proprietary selective preparation if antibodies for salmonella. This unique combination makes it one of the most sensitive and specific tests for salmonella.

1-2 Test ® For Salmonella Single test format – 1 test per device 2 Enrichment protocols Low microbial / Processed foods Aerobic plate count <10,000 CFU/g Product exposed to a processing step that reduces and controls bioburden (drying, freezing, cooking, etc…) High microbial / Raw foods Aerobic plate count >10,000 CFU/g Products possess inherent antimicrobial or bacteriostatic properties (spices) The 1-2 Test is the most widely used test for Salmonella screening in the world. This patented single-use unit is completeyl self-contained. The 1-2 test uses a unique combination of a built-in selective enrichment, immunodiffusion and a proprietary selective preparation if antibodies for salmonella. This unique combination makes it one of the most sensitive and specific tests for salmonella.

Enrichment Protocols 1-2 Test Processed / Low Microbial Foods PRE-ENRICHMENT Time to results: 36 – 54 hours BPW or Lactose broth @ 35 oC 24 ± 2h Transfer to 1-2 Test @ 35°C 14 – 30 hours Raw / High Microbial Foods SELECTIVE ENRICHMENT Time to results: 44 – 62 hours PRE-ENRICHMENT Iodine activated TBG @ 42 o C 6-8 h BPW or Lactose broth @ 35 oC 24 ± 2h Transfer to 1-2 Test @ 35°C 14 – 30 hours

1-2 Test - Procedure Position 1-2 Test unit with black cap up. Remove cap. Add 1 drop of Reagent #1 (Iodine-Iodide Solution) to TT Broth. Replace cap and gently shake unit to mix.

1-2 Test - Procedure Reposition 1-2 Test unit with black cap up. Remove plug with sterile forceps and discard it. This will allow communication between the two chambers.

1-2 Test - Procedure Transfer 0.1 mL sample into inoculation chamber For raw foods, transfer 1.5 mL of sample/TT mixture to empty inoculation chamber (side-port)

1-2 Test - Procedure Position 1-2 Test unit with white cap up and remove cap. Snip off tip of gel void former with shears and discard tip.

1-2 Test - Procedure Add 1 drop of Reagent #2 (Antibody preparation). Replace white cap. Incubate.

1-2 Test - Incubation 1-2 Test should be incubated with white cap up. Use the adequate incubation temperature: 35 °C Duration: 14 - 30 hours Could be incubated individually or while held in incubation tray

1-2 Test ® Advanced Science. Simple to Use. Motility Chamber Inoculation Chamber Diffusion of 1-2 Test antibody Antibody well Immunoband Movement of Salmonella As the Salmonella encounters the diffusing antibodies they are immobilized. A characteristic three-dimensional Immunoband will be formed if the test is positive. Results are read visually. Inoculation Chamber: enriched sample for analysis Motility Chamber: semi solid Agar based motility medium Antibody well: Salmonella specific antibodies Salmonella, if present, moves through the selective medium into the motility chamber. Antibody diffuses into the motility chamber in a radial pattern.

1-2 Test - Reading Results A positive test is indicated by the presence of a white band that is U-shaped or meniscus-shaped Immunoband is 3-dimensional and is seen in all planes while rotating 1-2 Test unit. The shape of the immunoband may vary from strain to strain. Refer to directions for use for other examples.

1-2 Test – Performance Statistics AOAC® Official Method of Analysis 989.13 Sensitivity: 98.2% Specificity: 98.8% False Negative Rate: 1.8% False Positive Rate: 1.2%

1-2 Test Objection Only detects motile Salmonella Motility is a key criteria used to define the genus Salmonella (Edward’s and Ewing’s Identification of Enterobacteriaceae, 4th ed. 1986) There are only 2 non-motile species of Salmonella, host adapted to poultry with very low virulence to humans (S. pullorum and S. gallinarum) 9 studies of the presence and distribution of Salmonella in food, feed, and dairy products – 183 species isolated with only 1 non-motile (S. pullorum from poultry facility environmental samples)

1-2 Test - Benefits Labor Savings Minimal hands-on time Processed foods- Lactose enrichment & transfer to 1-2 test Raw foods – Single selective enrichment (TT) Reduced media preparation Speed Raw – 44 hours (2 days) Processed – 36 hours (next day) Ease of use Least complicated Salmonella test on the market Easy to interpret