Listeria is widely found in nature, including soil, rivers, and vegetables. Since they are more resistant to heat, salt, and acidic environments compared to other bacteria and can even grow in refrigerators 1), they may cause food intoxication at food distribution levels when they grows to a specific infectious count.
The epidemic of infection with Listeria through foods including milk, cheese, vegetables, and meat has been reported around the world. In the United States, it is estimated that about 2,500 people are infected and 500 die every year 2). Although no food intoxication by Listeria has been reported in Japan, there is a possibility it can occur any time as the food contamination rate in Japan is almost the same as that in Western countries 3,4).
This kit is designed to detect Listeria in food by immuno-chromatographic technique and provides test results in a short time with a simple method.
To detect Listeria in food
Note 1: Since this kit is intended to detect Listeria, it cannot differentiate Listeria monocytogenes from other listerial bacteria.
1. This product does not need skill because it can be handled only with a one-step procedure.
2. It rapidly provides a test result.
3. It needs no specific detector.
A: Test plate: 5 tests×4
B: Instruction manual
2) Components and volume (per test)
(1) Anti-Listeria polyclonal antibody (rabbit) 0.5 μg
(2) Gold colloid labeled anti-Listeria polyclonal antibody (rabbit) 0.25 μg
(3) Anti-rabbit immunoglobulin polyclonal antibody (goat) 0.25 μg
1) Illustration of Test plate
a. Sample dropping part (Take care not to touch it with your hands)
b. Sample containing part
c. Spread area (Take care not to touch it with your hands or damage it)
d. Absorption pad
e. Measurement item entering area
f. Test line appearance position (about 30 mm from sample dropping part)
g. Control line appearance position (about 38 mm from sample dropping part)
2) Principle of assay
When a sample solution containing Listeria (1) is dropped at the sample dropping part of the test plate, the gold colloid labeled anti-Listeria antibody (2) is dissolved to form complexes with Listeria in the sample solution. The complexes migrate across the spread part by capillarity and are then trapped by the anti-Listeria antibody (3) fixed at the test line appearance position to develop a reddish purple line by the gold colloid. Thus, whether the sample solution contains Listeria or not can be determined by visually inspecting the test line appearance position.
Irrespective of the presence/absence of Listeria in the sample solution, the excess gold colloid labeled antibody further migrates across the spread area and is then trapped by the anti-rabbit immunoglobulin antibody (4) fixed at the control line appearance position to develop a reddish purple line. It can be confirmed that the sample solution has migrated across the spread area by the development of the line.
*: The preparation of the sample solution is based on ISO11290-1 5)
1) Necessary apparatus and equipment
Stomacher bag (that with a filter is recommended), stomacher, incubator (30°C and 35 to 37°C), autoclave, enrichment medium, thermostatic bath (that for which temperature can be set at 80°C or higher), and glass test tubes, etc.
2) Sample preparation
(1) Collect at least 200 g (or 200 ml) of the food to be tested.
(2) When the food is solid, collect small samples from as many sites as possible to make up a soli sample of 25g. When the food is liquid, stir it well before collecting a sample of 25 ml. For wipe test, use the gauze, etc. used for wiping as a sample or extract it in a small volume of dilution water and use the water as a sample.
3) Enrichment culture
(1) Add the sample to the primary enrichment medium (half Fraser culture medium) at a volume that is nine times as much as the sample. Treat it with a stomacher for an appropriate time depending on the sample and culture it at 30°C for 24 ± 2 hours.
(2) Inoculate 0.1 ml of the primary enrichment culture solution to 10 ml of the secondary enrichment medium (Fraser culture medium) and culture it at 35 to 37°C for 24 ± 2 hours.
Note1: Culture the sample in an EB culture medium at 30°C for 48 hours if the enrichment culture is performed according to the official inspection method in Japan 6,7). A UVM culture medium may also be used for culturing the sample when the sample is not milk or a milk product.
4) Sterilization operation
(1) After the culture is completed, remove the stomacher bag from the incubator and gently stir the culture solution with care not to splash it.
(2) Dispense 5 ml of the culture solution to a glass test tube with a sterilized pipette. Heat it at 80°C for 20 minutes and then use it as the sample solution.
Note1: Keep the remaining culture solution safely without sterilizing it until the end of the test because it may be used for confirming the test result of the kit.
1) Test procedure of NH IMMUNOCHROMATO Listeria
(1) Allow test plates to room temperature without removing them from the aluminum pouch and take out a necessary number of the test plates from the pouch immediately before use.
(2) Write the sample name or specimen number on the absorption pad of each test plate using a felt pen.
(3) Place the test plate on a horizontal plane and drop 100 μL of the sample solution onto the sample dropping part (see the following left figure). Alternatively, dispense 150 μL of the sample solution into a test tube and place the test plate into the test tube so that the sample dropping part will be immersed in the sample solution (see the following right figure).
(4) Allow the test plate to stand for 15 minutes and visually determine the result.
Note1: Since moisture may adversely affect the performance of the test plate, remove it from the aluminum pouch after it is allowed to room temperature.
Note2: Return any unused test plate into the plastic pouch with a desiccating agent and put the pouch into the aluminum pouch. Keep the aluminum pouch in a refrigerator.
Note3: Take care not to touch or damage the sample dropping part and spread area of the test plate. Hold the test plate by the absorption pad.
Note4: Use only a sterilized pipette or tip to drop or dispense the sample solution. Do not use one pipette or tip for more than one sample solution.
Note5: Take care not to make the sample solution overflow from the test plate when dropping the sample solution. Take appropriate measures for preventing the overflow, such as dividing the solution into two portions and dropping them separately, as required.
Note6: To prevent infection, spread a plastic wrap or equivalent and place a test plate on it when you choose to perform the test by dropping the sample solution onto the test plate.
2) Determination of result
(1) Determine the sample as positive when a reddish purple line is observed at both the test line appearance and control line appearance positions 15 minutes after the test is started. When no or only a light reddish purple line is observed at the test line appearance position at that time, observe the position again 60 minutes after the test is started.
(2) Determine the sample as negative when a reddish purple line is not observed at the test line appearance position, but is observed only at the control line appearance position.
(3) When no reddish purple line is observed at the control line appearance position, repeat the test, irrespective of the presence/absence of a reddish purple line at the test line appearance position because the spread of the sample solution may have been abnormal.
Note1: For the sample determined as positive by this kit, confirm the result with other test methods, such as the official inspection method. The sample cultured in the enrichment medium for this kit can be used for the confirmation tests, such as the official inspection method.
NH Immunochromato Listeria
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.