MFLP-95 The 20 Hour Reveal Method for Detecting Escherichia coli O157_H7 from Foods and Environmental Samples
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B429741B802947538166FE78EB1E00D8 |
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文件大小(MB)： |
0.03 |
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页数： |
7 |
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文件格式： |
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日期： |
2012-3-2 |
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Published on the Food Directorate’s (Health Canada) website at http://www.hc-sc.gc.ca/food-aliment.,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-95,September 2000,THE 20 HOUR REVEAL METHOD FOR DETECTING ESCHERICHIA COLI O157:H7,FROM FOODS AND ENVIRONMENTAL SAMPLES.,1. APPLICATION,The method is applicable to the detection of Escherichia coli O157:H7 from foods (such as apple cider,raw beef, and vegetables), and environmental swabs to determine compliance with Sections 4 and 7 of,the Food and Drugs Act.,2. DESCRIPTION,The method has been shown to produce satisfactory results with artificially contaminated foods and,naturally contaminated raw ground beef in AOAC studies (8.1) including apple cider, lettuce rinse, raw,beef cubes, raw ground beef, and environmental swabs . This method can be used successfully for the,detection of E. coli O157:H7 in other foods and samples.,3. PRINCIPLE,Following selective enrichment in either modified trypticase soy broth with novobiocin or modified EC broth,with novobiocin, a portion of the sample enrichment is placed into the sample port of the Reveal Device,initiating flow. The Reveal Device 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. 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 test completion and flow is indicated by a control line which forms,further up in the test window and verifies a valid test run. Absence of a control line invalidates the test.,Record results at 15 minutes incubation time.,4. DEFINITION OF TERMS,See Appendix A of Volume 3, items 1,2,3,4.,5. COLLECTION OF SAMPLES,See Appendix B of Volume 3.,6. MATERIALS AND SPECIAL EQUIPMENT,1) Reveal Device (Neogen Corporation, phone: 1-800-234-5333, Fax: (517) 372-0108.,2) Modified trypticase soy broth with novobiocin (base is commercially available; Acumedia, Baltimore,MD),3) Modified E. coli broth with novobiocin (base is commercially available; Acumedia, Baltimore, MD),4) Biosynth BCM O157:H7+ chromogenic agar (Biosynth, Naperville, IL),5) Hemorrhagic coli agar,6) MacConkey sorbitol agar (Acumedia, Baltimore, MD),MFLP-95,- 2 - September 2000,7) mHC agar or TCCSMAC agar (see MFLP-80),8) Butterfield’s phosphate buffer (Fisher Scientific) or peptone water 0.1%,9) Incubators capable of maintaining 36EC.,10) Incubators capable of maintaining 42EC.,NOTE: It is the responsibility of each laboratory to ensure that the temperature of the incubators or water,baths are maintained at the recommended temperatures. Where 36EC is recommended in text of,the method the incubator may be at 36 +/-1.0E C. Similarly, lower temperatures of 30 or 25 may be,+/- 1.0EC. However, where higher temperatures are recommended, such as 43 or 45.5EC, it is,imperative that the incubators or water baths be maintained within 0.5EC due to potential lethality,of higher temperatures on the microorganism being isolated.,11) ?Stomacher ?Colworth or equivalent,12) ?Stomacher ?bags,13) Micropipette (200 μl) and sterile tips,14) Control cultures,E. coli O157:H7 ATCC # 35150; ATCC # 43895,7. PROCEDURE,Each sample unit may be analyzed individually or the analytical units may be combined. Carry out the test,in accordance with the following instructions:,7.1 Handling of Sample Units,7.1.1 In the laboratory prior to analysis, keep sample units refrigerated (0-5EC) or frozen,depending on the nature of the product. Thaw frozen samples in a refrigerator, or under,time and temperature conditions which prevent microbial growth or death.,7.1.2 Analyze sample units as soon as possible after their receipt in the laboratory,7.2 Preparation for Analysis,7.2.1 Have ready modified trypticase soy broth or modified E. coli medium and prepared,novobiocin.,7.2.2 Clean the surface of the working area with a suitable disinfectant.,7.3 Preparation of Sample,7.3.1 To ensure a truly representative analytical unit agitate liquids or free flowing materials until,the contents are homogeneous. If a sample unit is a solid, obtain the analytical unit by,taking a portion from several locations within the sample unit. To reduce the workload, the,analytical units may be combined for analysis. It is recommended that a composite contain,not more than 500g.,7.3.2 Lettuce and other vegetables: aseptically obtain 50……
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