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One important limitation to metabolic tests is that they almost always require the growth of the microbe in some medium and this takes at least 12-24 hours before a clear result is obtained. Faster results would speed diagnosis. A second limitation to metabolic tests is that a specific isolate under testing may have a mutation such that it is incapable of performing a metabolic conversion where the normal isolate of that species is proficient. For example, most strains of E. coli are capable of using lactose as a carbon source, but isolates of E. coli have been found that cannot utilize lactose. Finally, to clearly identify an isolate, it is often necessary to perform numerous tests. The preparation and use of all this media is time consuming and expensive.

Because of the limitations of metabolic tests, considerable effort has been made to find accurate, simple, and rapid alternatives. This research is starting to bear fruit, with the most popular methods being antibody tests and DNA methods, especially Figure 6-13 shows the steps of the TECRA reaction.

Here we present the TECRA reaction, explaining the theory behind how it works and a typical protocol for its use. The protocol was adapted from the technical material provided by BioTrace International.

This kit will be used for various experiments depending upon time and funding. The protocol is the same for both after the initial enrichment steps. These initial enrichment steps will be done according the FDA approved BAM. The majority of this section is modified from the instructions included with the TECRA kit.

1. High affinity "capture" antibodies specific for the test microbe have been adsorbed on the surface of the Removawells.
2. If the test microbes antigens are present in the sample, they are captured by the antibodies. All other materials in the sample are washed away.
3. The sandwich is completed by the addition of enzyme-labeled antibodies (Conjugate) specific for the test microbe.
4. The presence of the test microbe is indicated when the bound conjugate converts the Substrate to a green color. Alternatively, test microbe, no green color.

Intended use

The TECRA® Visual Immunoassay (VIA) is a rapid and specific screening test for the in vitro detection of the species of interest in food, food-related and environmental samples after enrichment.

Following enrichment, at least 1 CFU of the species of interest in 25 grams of sample can be detected. Presumptive positives should be confirmed by standard methods. This is especially important in situations such as product recalls.

The TECRA® VIA utilizes antibodies which recognize all tested strains of the species of interest. This method has been shown to be at least as sensitive as standard culture and plating techniques and has virtually no cross-reactivity with other types of organisms.

The TECRA® VIA is an Enzyme-linked Immunosorbent Assay (ELISA) performed in a "sandwich" configuration.

Performing the Immunoassay:

1. Ensure all kit components are at room temperature (20-25°C) before use.
2. Transfer 0.5 ml of each enrichment into a labeled microfuge tube.
3. Heat the tube for 15 minutes at 100 °C in the heating block.
4. Open the pouch and remove the required number of wells from the sealing film, allowing one well for each sample (a positive and negative control are also necessary for comparison). Press the wells firmly into place in the holder.
5. Using a new pipette tip for each sample, transfer 200 µl aliquots of the samples into individual wells. Cover the wells with plastic cling wrap film and incubate for 30 minutes at 35-37 °C
6. NOTE: Thorough washing in these next steps is a critical step and is essential for a clear interpretation of the results.
7. Dump out the liquid in the Removawell. Remove residual liquid by striking the holder firmly several times face down on a thick pile of absorbent paper towels. This is important for effective removal of sample residue.
8. Completely fill each well with wash solution, taking care not to trap air bubbles in the bottom of the wells. Wash and completely empty the wells a total of 3 times as outlined above.
9. Ensure the Removawells are empty before proceeding.
10. Add 200 µl of Conjugate to each well. Cover the holder with plastic cling wrap film and incubate for 30 minutes at 35-37°C
11. Empty the wells and wash them thoroughly a total of FOUR times (not three) using the sequence previously described in step 7
12. Ensure the Removawells are empty before proceeding.
13. Add 200 ml of Substrate to each well. Incubate at 20-25°C (room temperature). Color development tends to concentrate around the sides of the wells. Tap the holder gently to distribute the color evenly before reading the result.
14. Incubate for 10 minutes, then read the results.
15. Results can be read visually using the Color Card provided OR with a plate reader.
16. If using the card, compare your sample to the card.
17. If using a plate reader, perform steps 18-23
18. The absorbance of the samples can be read at 414+10 nm using the plate reader. We will use the 415 nm filter.
19. Dual wavelength instruments should be blanked against air and the second "reference" wavelength should be set at 490+10nm.
20. After 10 minutes, add 200 ml of Stop Solution to each well. Tap the sides of the holder gently to mix the contents, then read the result.

For the test to be valid:

• The positive control must have an absorbance of at least 0.3 and the negative control must have an absorbance of less than 0.2.
• A sample is considered negative when the assay has proved valid and the sample well has a reading of less that 0.3.
• A sample is considered positive when the assay has proven valid and the sample well has a reading greater that or equal to 0.3.

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