Tests for pathogenic Escherichia coli in foodstuffs
Tests for pathogenic Escherichia coli in foodstuffs
The test of pathogenic bacteria in food mainly includes the test of various pathogenic bacteria in food raw materials and food, such as Staphylococcus aureus, Streptococcus haemolyticus, Salmonella, Shigella, Vibrio parahaemolyticus, Clostridium botulinum and toxin, Pasteurella, and other 19 kinds of pathogenic bacteria. Only the test for Escherichia coli is presented here in order to grasp the principles and methods of pathogenic Escherichia coli testing.
Operation method
double antibody sandwich method
Principle
Under normal circumstances, Escherichia coli is not pathogenic and can synthesize vitamins B and K and produce colistin, which is beneficial to the organism. However, when the body's resistance is reduced or Escherichia coli invades extraintestinal tissues or organs, it can cause extraintestinal infections as a conditional pathogen. Some serotypes can cause intestinal infections, and there are four known categories of Escherichia coli that cause pathogenicity, namely, enterotoxin-producing Escherichia coli, hemorrhagic Escherichia coli, intestinal invasive Escherichia coli, and intestinal pathogenic Escherichia coli, the latter of which mainly causes diarrhea in newborns. Bacterial cattle and pigs are an important cause of food poisoning caused by the spread of the bacteria, and human carriers can also contaminate food and cause poisoning.
Materials and Instruments
Escherichia coli Standard Strain Food Sample White Rat Escherichia coli Diagnostic Serum Enterotoxin-producing Escherichia coli Diagnostic Serum Enterotoxin-producing Escherichia coli LT and ST Enzymatic Diagnostic Kits Anti-LT Antitoxin Move I. Enrichment For more product details, please visit Aladdin Scientific website.
Polymyxin B Paper Sheets Thiomersal Solution Evans Blue Solution Gram Stain Broth MacConkey Agar Erythromycin Blue Agar Triple Sugar Iron Agar Kirschmann's Double Sugar Iron Agar Sugar Hairpin Tubes Lysine Decarboxylase Test Medium Urea Agar Potassium Cyanide Medium Protein Chen Water Indocyanine Reagent Semi-Solid Agar Honda's Toxin-Producing Broth E1ek's Medium Oxidase Reagent
Balance Homogenizer Mantle Warmer Water Bath Microscope Centrifuge Enzyme Labeler Bacterial Concentration Turbidimeter Wide Mouth Flask Triangular Flask Petri dish Test Tubes Straws Rubber Nipples Slides Alcohol Lamp Metal Spoon Glass Rods Nickel Chromium Wire Inoculation Rods Test Tube Racks Test Tube Baskets Refrigerator Syringe Knife Scissors Tweezers Nitric Acid Filter Filter Filter Filter Films
Samples should be examined as soon as possible after collection. Weigh 25 g of the test sample aseptically, add it to 225 mL of nutrient broth, and break it up with a homogenizer for 1 min or grind it up with a milk bowl and sterilized sand. Remove the appropriate amount, inoculate the lactose bile salt medium for the determination of coliform MPN, and transfer the rest into a 500 mL wide-mouth bottle and incubate at 36 soil 1 ℃ for 6 h. Pick 1 ring and inoculate it into 1 tube of 30 mL Enterobacteriaceae enrichment meat field and incubate it at 42 ℃ for 18 h. The sample was then transferred into a 500 mL wide-mouth bottle and incubated at 36 soil 1 ℃ for 6 h.
II. Separation
Lactose fermentation positive lactose bile salt fermentation tubes and bacteriophage increase inoculation McConkey or Erythromycin agar plate; serious contamination of the test samples, the test sample homogenate can be directly inoculated with McConkey or Erythromycin plate, incubated at 36 soil 1 ℃ for 18 h a 24 h, observe the colonies. Not only should we pay attention to the lactose fermentation colonies, but also pay attention to lactose non-fermentation and delayed fermentation colonies.
III. Biochemical tests
1. Several colonies were picked directly from the identification plates and inoculated with trisaccharide iron (TSl) or kirschner's disaccharide iron agar (KI), respectively. At the same time, these cultures were inoculated with peptone water, semi-solid, pH 7.2 urea, agar, KCN broth and lysine decarboxylase test medium, respectively. All the above cultures were incubated at 36 °C overnight.
2. TSI slant acid-producing or non-acid-producing, bottom acid-producing, H2S-negative, KCN-negative and urea-negative cultures are Escherichia coli. cultures that are not acid-producing on the bottom of the TSI slant or that are positive for any one of the following: H2S, KCN, or urea, are not Escherichia coli. Perform oxidase test or Gram stain microscopy if necessary.
IV. Serological tests
1. Hypothetical tests
Agar cultures confirmed as E. coli by biochemical tests were picked and slide agglutination tests were done with polyvalent O sera of pathogenic E. coli, invasive E. coli vaccine, enterotoxin-producing E. coli, and hemorrhagic E. coli vaccine O157, sera. When agglutinating with a particular polyvalent O serum, the test is then performed with the monovalent O serum contained in that polyvalent serum. If a strong agglutination reaction is shown with one of the monovalent O sera, the test is assumed to be positive.
2. Confirmation experiments
An O antigen suspension was prepared and diluted to a concentration comparable to that of a MacFarland?No. 3 turbidimetric tube. O serum with original potency of 1:160-1:320 was diluted with 0.% saline to 1:40. Diluted serum and antigen suspension were mixed in equal amounts in 10 mm×75 mm tubes for tube agglutination test. After mixing, put it in 50 ℃ water bath box and observe the result after 16 h. If agglutination occurs, it can be confirmed. If agglutination occurs, it can be confirmed as the O antigen.
V. Enterotoxin test
1. Enzyme-linked immunosorbent assay (ELISA) to detect LT and ST
(1) Toxin-producing cultures
The test strain and the positive and negative control strains were inoculated into 0.6 mLCAYE medium, and incubated overnight at 37 ℃ with shaking. Add 20000 IU/ml polymyxin B 0.05 mL, incubate at 37 ℃ for 1h, centrifuge at 4000 r/min for 15 min, separate the supernatant, add 0.1% thiomersal 0.05 ml, and store at 4 ℃ for use.
(2) LT detection method (double antibody sandwich method)
Inclusion: Firstly, take out the LT antibody tube for inclusion in the CT and ST enzyme diagnostic kit for enterotoxin-producing Escherichia coli, add 0.5 ml of inclusion solution, mix it well, then suck out all of it into 3.6 ml of inclusion solution and mix it well, and then add it into 40-well polystyrene hard reaction plate in the amount of 100 μl per well, and the first well was left empty as a control, and then leave the plate in a wet box in a refrigerator at 4 ℃ overnight.
Wash the plate: Shake off the solution in the plate, wash it 3 times with washing solution I, shake off the liquid, turn over the reaction plate and tap it on absorbent paper to remove the residual liquid in the wells.
Sequestration: Add 100 μL of sequestration solution to each well and incubate for l h in a 37 ℃ water bath.
Plate washing: 3 washes with washing solution II, same operation as above.
Add samples: Add 100 μl of virulence-producing culture medium of various test strains to each well, and incubate for l h at 37 ℃ in a water bath.
Plate washing: 3 washes with washing solution II, same operation as above.
Add enzyme label antibody: first add 0.5 ml dilution solution in enzyme label LT antibody tube, mix well and then all pipette out of 3.6 ml dilution solution to mix, add 100 μl per well, 37 ℃ water bath in 1 h.
Plate washing: 3 washes with washing solution II, same operation as above.
Enzyme substrate reaction: 100 μl of substrate solution was added to each well (including the first well), and the final solution was added 50 μl after 5 min~10 min of light protection at room temperature.
Determination of results: The absorbance 0D value was measured by enzyme marker at the wavelength of 492 nm, and the OD value of the specimen to be tested was more than 3 times larger than that of the negative control as positive, and the color of the visual inspection was barrel yellow or significantly higher than that of the negative control as positive.
(3) ST detection method (antigen competition method)
Inclusion: First add 0.5 ml of inclusion solution to the ST antigen tube for inclusion, mix well, then pipette all the solution out of 1.6 ml of inclusion solution and mix well, and add it into 40-well polystyrene soft reaction plate at 50 μl per well. After adding the liquid, gently knock the plate to make the liquid cover the bottom of the wells. The first well was left empty as control and placed in a wet box at 4 ℃ overnight.
Plate washing: 3 washes with washing solution I, same operation as above.
Closure: Add 100 μl of closure solution to each well, 37 ℃ water bath than.
Plate washing: 3 washes with washing solution II, same operation as above.
Add samples and ST monoclonal antibody: Add 50 μl of virus-producing culture medium of each test strain and 50 μl of diluted ST monoclonal antibody to each well (add 0.5 mL of diluent to the ST monoclonal antibody tube, mix well and then aspirate all of them out of 1.6 ml of diluent, and mix them), and then incubate in a water bath at 37 ℃ for 1 h. The sample should be mixed with the diluted ST monoclonal antibody tube for 1 hour.
Plate washing: 3 washes with washing solution II, same operation as above.
Add enzyme-labeled rabbit anti-mouse 1 g complex: first add 0.5 mL of diluent to the enzyme-labeled anti-mouse 1 g complex tube, mix well and then pipette all out of 3.6 mL of diluent and mix well, add 100 μl to each well, and then water bath for 1 h at 37 ℃.
Plate washing: 3 washes with washing solution II, same operation as above.
Enzyme substrate reaction: add 100 μl of substrate solution to each well (including the first well), avoid light for 5 min~10 min at room temperature, then add 50 μl of termination solution.
Determination of results: The absorbance OD value was measured at 492 nm with an enzyme marker; visually colorless or significantly lighter than the negative control was considered positive.
2. Two-way agar diffusion test for LT
The examined strains were inoculated on E1ek's medium in a five-point loop. With the same operation, two copies were made and incubated at 36 ℃ for 48 h. A polymyxin B paper sheet was placed on each moss, and the plate was incubated at 36 ℃ for 5 h-6 h to allow the enterotoxin to penetrate into the agar, and in the center of the five-point circular mosses, 5 mm from each point, a round hole of 4 mm in diameter was dug and cushioned with a drop of agar. A drop of 30 μl of LT antitoxin was added to the central hole of the plate, and known LT-producing and non-toxin-producing bacteria were used as controls, and the results were observed after 15 h to 20 h. The results of the experiment were observed in the center of the plate. The presence of a white precipitation band between the plaque and the antitoxin wells was considered positive, and the absence of a precipitation band was considered negative.
3. Gavage test for detection of ST in suckling mice
The tested strain was inoculated into Honda's poison broth, incubated at 36 ℃ for 24 h, centrifuged at 3000 r/min for 30 min, and the supernatant was filtered through a membrane filter, heated at 60 ℃ for 30 min, and 2% Evans blue solution was added into each mL of the filtrate 0.02 mL. The filtrate was injected into the stomachs of 1-day-old and 4-day-old suckling rats with a plastic tubule for 0.1 mL, and 3~4 rats were inoculated at the same time. After fasting for 3h～4h and anesthetized with trichloromethane, all the intestinal tubes were removed, and the weight of the intestinal tubes (including the fluid) and the remaining body weight were weighed. The ratio of the weight of the intestinal tubes to the remaining body weight was greater than 0.09 as positive, and 0.07-0.09 as suspicious.