Food poisoning caused by Diarrheagenic Escherichia coli is caused by the growth of E.coli taken into the intestinal tract though the ingestion of food. These Diarrheagenic E.coli can be broadlyified into five types.1) Among these, Enterohemorrhagic E.coli (EHEC) causes hemorrhagic colitis through the action of the verotoxin produced; it can also cause hemolytic uremic syndrome and encephalopathy, which can be fatal. Many cases of food poisoning by EHEC have occurred, so EHEC is viewed as most dangerous among Diarrheagenic E.coli from the standpoint of food hygiene. In addition to E. coli O157, other serotypes have been detected, such as O26, O111, O118, O103, and O121. In Japan, however, most of EHEC are reported to be of E.coli O157.2)
These products are kit for detecting E. coli in foods by immunochromatography. Tests can be conducted rapidly and simply by means of the kit.
(1) The simple one-step operation of the kit.
(2) The test gives rapid results.
(3) There is no need for special test equipment.
A: Test plate 5-test × 4 packs
B: Instruction manual 1 sheet
2) Ingredients and Quantity
A: Anti-E.coli polyclonal antibody (rabbit) 0.5 g
B: Anti-E.coli polyclonal antibody labeled with colloidal gold (goat) 0.13 g
C: Anti-goat immunoglobulin polyclonal antibody (rabbit) 0.25 g
1) Illustration of Test plate
2) Principle of assay
When a sample solution is dropped onto the test sample drop section of the test plate, the gold colloid-labeled anti-E. coli polycolonal antibody (2) in the test sample-containing section dissolves and forms complexes with E. coli (1). These complexes move to the expanded section by capillary attraction and are trapped by the anti-E. coli antibody (3) that is fixed in the test line appearance position. This results in the appearance of a reddish purple line of gold colloid. This reddish purple line can be detected by visual inspection and used to judge the presence or absence of E. coli in the test solution. The excess gold-labeled antibodies, regardless of the presence or absence of E. coli in the test solution, travel further through the expanded section and are trapped by the anti-goat immunoglobulin rabbit antibody(4) fixed at the control line appearance position, where they form a second reddish purple line. The presence of this line indicates that the test solution has reached the expanded section.
The method for preparing a sample solution is described in accordance with the Japanese official testing method.3),4)
1) Required Equipment and Instruments
Stomacher bag (preferably with a filter), stomacher, incubator, autoclave, culture medium, etc
2) Preparation of Test Samples
(1) Take a test sample of more than 200g of the food under test. In cases where surface contamination is suspected, the sample is taken by scraping off 300500cm2 of the surface to a thickness of 0.20.3mm.
(2) Chop and mix the whole sample collected. Weigh 25g of the sample into the stomacher bag and use this as the test specimen
3) Sample enrichment
(1) Add 225 mL of mEC broth with novobiocin to the 25g specimen in tomacher bag and homogenize with a stomacher for 1 minute.
(2) Incubate the specimen in the stomacher bag at 42°C for 1824 hours.
Note1: When commercial mEC culture medium that does not contain novobiocin is used, sterilize the medium by autoclaving and cool it. Then, sterilize a 4 mg/mL solution of novobiocin by filtration and add 5 mL of the solution to 1 L of mEC culture medium to give a final concentration of 20 mg/L.
Note2: Instead of the mEC broth with novobiocin, it is possible to use tryptosoya broth (TSB) or growth and proliferation retardant-added TSB (mTSB, TSB-CTV, or mTSB-VCC) selective enrichment broth.
(1) Remove the stomacher bag from the incubator after 18-24 hours. Gently mix the contents of the stomacher bag using a side-to-side motion, taking care not to splash it.
(2) Transfer about 5 mL of the culture solution into a glass test tube by using a sterilized pipette, and sterilize the solution in an autoclave for 20 minutes at 121 °C.
(3) Remove the tube from autoclave and allow the tube to cool to room temperature to prepare the sample solution.
Note1: The kit can also detect viable E.coli, but tests with a sterilized culture solution are recommended to ensure the safety of the operator.
Note2: Because the remainder of the culture solution might be required for use in confirmatory tests following those conducted with the kit, do not sterilize it and retain it until all the tests have been completed.
1) NH Immunochromato E.coli Test Procedures
(1) Bring the test plates contained in the aluminum pouch to room temperature and remove from the pouch as necessary immediately before use.
(2) With an oil-based marker pen, write the name of the test sample or the number of the subject under test on the absorbent pad of the test plate removed from the bag.
(3) Place the test plate carefully on the flat stand and drop a 100 uL-portion of the test solution onto the test sample drop section (see the figure on the left). Otherwise, dispense a 150 L portion of the test solution into a test tube and attach the test plate to the test tube so that the test sample drop section of the test plate is immersed in the test solution (see the figure on the right).
(4) Allow the test plate to stand undisturbed for 15 minutes and then visually judge the presence or absence of E. coli in the solution.
Note1: Do not remove the test plate from the aluminum pouch until it has returned to room temperature, otherwise incorrect test results may be obtained as a result of moisture absorption.
Note2: Keep test plates that are not in use in a vinyl pouch containing desiccants and store this in an aluminum pouch in a refrigerator.
Note3: Be careful not to scratch the test sample drop section or expanded section and do not touch them with your hands. When handling the test plate, make sure that you hold the absorbent pad.
Note4: Make sure that you use a sterilized pipette or chip to drop or dispense the sample solution. Change the pipette or chip for every test solution.
Note5: Make sure that the 150-L portion of test solution does not overflow the test plate when dropping it. If necessary, drop the solution in two or more portions.
Note6: To prevent infection of the operator, it is recommended that testing be performed with the test plate. A wrap should be placed under the plate. when dropping the test solution.
2) Judgment of the Test Results
(1) The test results is judged as positive when a reddish purple line is observed at the test line appearance position and at the control line appearance position 15 minutes after the start of the test. When a reddish purple line is not observed on the test line appearance position 15 minutes after the start of the test or the test line is only slightly colored, recheck the results 60 minutes after the start of testing.
(2) Judge the test results as negative when no reddish purple line is observed at the test line appearance position, but a line is observed at the control line appearance position.
(3) Retest in cases where no reddish purple line is observed at the control appearance position, regardless of the presence or absence of a line at the test line appearance position. It is likely that there is something abnormal in the development of the sample solution in such cases.
Note1: Ensure that confirmatory tests, in accordance with the official test method or other methods, are performed on specimens that are tested positive by the kit. The proliferated and cultivated samples used for testing with the kit can be used in confirmatory tests by the official or other test method.
NH Immunochromato O157
NH Immunochromato O26
NH Immunochromato O111
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.