AOAC Official Method_aoac17.4.01a
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46162997189D4716998D7FFD02E7F57D |
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0.06 |
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2 |
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日期： |
2024-7-30 |
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17.4.01A,AOAC Official Method 996.09,Escherichia coli O157:H7 in Selected Foods,Visual Immunoprecipitate Assay (VIP?),First Action 1996,Final Action 1999,(Applicable to detection of E. coli O157:H7 in dairy foods, meats,poultry products, fruits, nutmeats, seafood, pasta, and liquid eggs.),See Table 996.09 for the results of the interlaboratory study supporting,the acceptance of the method.,A. Principle,Proprietary antibodies, with high specificity to E. coli O157:H7,antigens, are bound to chromogenic carrier and, separately, to solid,support matrix. Reagents are incorporated in test units and produce,visually discernible reaction product in presence of E. coli,O157:H7. During initial hydration of test unit, E. coli O157:H7 reacts,with antibody-chromogen complex to form antigen-antibody,chromogen complex, which flows across lateral flow membrane,and is bound by antibody immobilized on membrane. Positive reaction,is indicated by presence of detection line positioned across,the solid support in test sample window. Proper test completion is,indicated by another line formed in test verification window. Absence,of line in test verification window invalidates the test.,B. Media and Reagents,(a) VIP unit..One per test sample. Available as VIP?Visual,Immunoprecipitate Assay for E. coli O157:H7 (EHEC) from Bio,Control Systems, Inc., 12822SE32nd St, Bellevue,WA98005, USA.,(b) Novobiocin solution..100 mg/mL. Dissolve 100 mg,novobiocin in 1.0 mL H2O. Filter sterilize using 0.45 mm or smaller,porosity filter attached to syringe. Solution is stable up to 60 days,when stored in dark bottle at 2.8°C.,(c) Modified trypticase soy broth with novobiocin,(mTSBN)..Mix 30.0 g trypticase soy broth (dehydrated), 1.5 g bile,salt No. 3, and 1.5 g K2HPO4 (anhydrous) in 1 L H2O. Autoclave,mixture and let cool to room temperature. Add 0.2 mL novobiocin,solution/L media, (b), just before preparation of test sample.,(d) Diagnostic reagents..Necessary for culture confirmation of,presumptive positive VIP tests. See chapter on Escherichia coli and,Coliform Bacteria in Bacteriological Analytical Manual, current,edition, AOAC INTERNATIONAL, Gaithersburg, MD 20877,USA; or Microbiology Laboratory Guidebook, U.S. Department of,Agriculture.Food Safety Inspection Service, Athens, GA 30604,USA.,(e) Triton X-100..Used for preparing viscous foods.,C. Apparatus,(a) Incubators..Maintaining 35.37°C and 42.43°C (used in,confirmation procedure only).,(b) Syringe..With 0.45 mm or smaller porosity filter.,(c) Micropipets..Accurately dispensing 0.1 mL.,(d) Steam bath..Used for steaming Triton X-100 when preparing,viscous foods.,(e) Top loading balance..For weighing test portions.,D. General Instructions,Store VIP units in foil pouch with desiccant at ambient temperature,in cool dark place. After use, discard units into appropriate,decontamination container and sterilize before disposal. Do not,reuse and do not use VIP units after expiration date.,Run positive and negative control cultures to become familiar,with interpretation of results.,E. Preparation of Test Sample,Enrichment..Aseptically weigh 25.0 g test portion into 225 mL,mTSBN, B(c). If larger test portion sizes are analyzed, proportionately,increase volume of mTSBN to maintain 1:9 dilution ratio. Mix,well. Incubate overnight (18.28 h) at 35.37°C.,If samples produce viscous liquid (i.e., powdered cheese, raw,meats), add ￡2.25 mL steamed (15 min) Triton X-100 per 225 mL,modified mTSBN prior to incubation.,F. VIP Procedure,Open sealed pouch and remove required number of VIP units. Use,1 unit/test sample. Do not reuse VIP units. Reseal unused VIP units,in pouch containing desiccant and store at ambient temperature in,cool dark place.,Gently shake incubated enrichment broth from E and let food particles,settle. Transfer 0.1 mL incubated enrichment broth to sample,well and incubate 10 min at ambient temperature. Proceed immediately,to G.,G. Reading and Interpreting Results,(Note: Examine device immediately after 10 min incubation. Otherwise,faint lines may develop because of nonspecific color development,which should be disregarded.),Examine VIP unit for the presence of distinct detection lines in,test sample and test verification windows. Lines should be dark,when contrasted with white background and should extend across,window. Intensity of test sample and test verification lines may differ.,Test is valid if line is present in test verification window.,Test sample is considered positive when distinct lines are present in,test and test verification windows. Test sample is considered negative,when control is valid and no line is seen in test sample window. If no,line is pre……
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