AOAC Official Method 2000.14 Reveal for E
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264C2FDE507F414FA341C5F3F39F83D0 |
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0.03 |
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2 |
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日期： |
2024-7-30 |
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17.4.07,AOAC Official Method 2000.14,Reveal for E. coli O157:H7 Test in Selected Foods,and Environmental Swabs,20-Hour Method,First Action 2000,(Applicable only to detection of E. coli O157:H7 in environmental,swabs of stainless steel, apple cider, iceberg lettuce rinse, raw,beef cubes, and raw ground beef.),See Table 2000.14 for the results of the interlaboratory study supporting,acceptance of the method.,A. Principle,Reveal system contains antibodies with high specificity to E. coli,O157:H7 antigens. These antibodies are bound to colloidal gold and,separately, to a solid support matrix.Aportion of the test enrichment,is placed into the sample port of the Reveal system initiating flow.,Any E. coli O157:H7 antigen present will bind to the,gold-conjugated antibodies forming an antigen–antibody–,chromogen complex. This complex flows across a lateral flow,membrane and is subsequently bound by antibody immobilized on,the membrane. This causes the gold conjugate to precipitate, forming,a visible line and indicating a positive reaction. Proper assay,completion and flow are indicated by a control line which forms,higher up in the device window, verifying a valid assay. Absence of,a control line invalidates the assay.,B. Media and Reagents,(a) Reveal device.—One assay per Reveal device. Available,from Neogen Corp., 620 Lesher Pl, Lansing, MI 48912.,(b) Butterfield’s phosphate buffer (BPB) diluent.—Mix 34.0 g,monopotassium phosphate in 500 mL distilled water. Adjust pH to,7.2 using 1M NaOH (40 g/L). Dilute to 1 L with distilled water. Autoclave,for 15 min at 121°C. Add 1.25 mL to sterile 1 L flask and dilute,to 1 L with sterile water.,(c) Novobiocin solution, 100 mg/mL.—Dissolve 100.0 mg,novobiocin in 1.0 mL water. Sterilize by filtration through 0.45 mm,filter attached to a syringe. This solution is stable for 60 days when,stored in dark bottle at 2–8°C.,(d) Modified Trypticase soy broth with novobiocin,(mTSB+n).—Mix 30.0 g Trypticase soy broth (Becton Dickenson,Cockeysville, MD), 1.5 g bile salt No. 3 (Becton Dickenson), and,1.5 g dipotassium phosphate in 1 L deionized water. Autoclave mixture,for 15 min at 121°C and let cool to room temperature. Add 0.2mL,novobiocin solution, (c), just before addition of test suspension.,(e) Modified E. coli broth with novobiocin (mEC+n).—Mix,20.0 g tryptone (Becton Dickenson), 1.5 g bile salt No. 3 (Becton,Dickenson), 5.0 g lactose, 4 g dipotassium phosphate, 1.5 g,monopotassium phosphate, and 5 g NaCl in 1Ldeionized water. Autoclave,mixture for 15 min at 121°C and let cool to room temperature.,Add 0.2mLnovobiocin solution, (c), just before addition of test,suspension.,(f) Diagnostic reagents.—Necessary for culture confirmation of,presumptive positive Reveal results as described in BAM or FSIS,Laboratory Communication No. 38.,(g) Hemorrhagic coli (HC) agar.—Prepare as follows: tryptone,20.0 g; bile salts No. 3, 1.12 g; NaCl, 5.0 g; sorbitol, 20.0 g;,4-methylumbelliferyl-b-D-glucuronide (MUG) reagent, 0.1 g;,bromocresol purple, 0.015 g; agar, 15.0 g; and distilled water, 1.0 L.,Dissolve ingredients in distilled water by heating with stirring. Autoclave,15 min at 121°C. Final pH 7.2 ± 0.2.,(h) Biosynth Chromogenic Medium (BCM) agar.—Available,from Biosynth International, Inc., 1665 W. Quincy Ave, Suite 155,Naperville, IL 60504, USA.,C. Apparatus,(a) Incubators.—Maintaining 35°–37°C and 43° ± 0.5EC.,(b) Syringe.—With 0.45 mm or smaller porosity filter.,(c) Pipettor.—Accurately dispensing 0.12 mL.,(d) Graduated cylinder.—250 mL capacity with 5 mL graduations.,(e) Stomacher bag.,D. Preparation of Test Suspension,Enrichment.—For environmental swabs of stainless steel, aseptically,transfer one swab into stomacher bag,C(e), containing 225mL,mTSB+n, B(d). Mix by hand-kneading and incubate 20 h at,35°–37°C. Cool to room temperature.,For apple cider, aseptically transfer 25 mL into 500 mL,screw-capped Erlenmeyer flask containing 225 mL mTSB+n, B(d).,Cap loosely, mix by shaking lightly, and incubate 20 h at 35°–37°C.,Cool to room temperature.,For iceberg lettuce, aseptically shred lettuce and transfer 50 g to,sterile bag. Add 50 mL BPB diluent, B(b), close bag and shake by,hand for 15 min. Aseptically decant 25 mL into 500 mL,screw-capped Erlenmeyer flask containing 225 mL mTSB+n, B(d).,Cap loosely, mix by shaking lightly, and incubate 20 h in air incubator,at 43° ± 0.5°C. Cool to room temperature.,For raw beef cubes and raw ground beef, aseptically weigh 25.0 g,into stomacher bag C(e) containing 225 mL mEC+n, B(e). Mix well,by hand-kneading and incubate 20 h at 35°–37°C. Cool to room temperature.,E. Assay Procedure,Open pouch containing one Reveal device, ……
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