MFLP-92 Detection of Escherichia coli O157 in Foods by Polymyxin-based Enzyme-linked Immunosorbent Assay
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4E09D557407F4FCCAB7857DCCC047848 |
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0.09 |
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5 |
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日期： |
2024-7-30 |
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Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-92,June 2005,HEALTH PRODUCTS AND FOOD BRANCH,OTTAWA,DETECTION OF ESCHERICHIA COLI O157 IN FOODS BY POLYMYXIN-BASED,ENZYME-LINKED IMMUNOSORBENT ASSAY,Burton Blais1*, Jessica Bosley1, Amalia Martinez-Perez1 and Donna Douey2,1Research and Development Section, Ottawa Laboratory Carling (OLC),Canadian Food Inspection Agency,Ottawa, Ontario,K1A 0C6,2National Laboratory Operations,Calgary Laboratory,3650 36th St. N.W.,Calgary, AB,T2L 2L1,* Principal contact:,E-mail: bblais@inspection.gc.ca,1. APPLICATION,The method is applicable to the the rapid detection of Escherichia coli O157 in enrichment broths from a,variety of foods and other samples prepared by the Health Canada Escherichia coli O157:H7 culture,technique, MFLP-80 (9.3). It can be used to determine compliance with Sections 4 and 7 of the Food and,Drug Act.,2. PRINCIPLE,After initial enrichment of samples in modified trypticase soy broth with novobiocin, Escherichia coli O157,lipopolysaccharide (LPS) antigens are extracted from enrichment broth cultures by heating in the presence,of sodium cholate. The extracted LPS antigens are detected by reaction with polymyxin-coated,microwells, followed by detection of the captured LPS antigens by sequential reactions with a,commercially available anti-E. coli O157 antibody-peroxidase conjugate and the chromogenic substrate,tertramethylbenzidine (TMB). The polymyxin-ELISA has been shown to be highly sensitive and specific,for E. coli strains bearing the O157 antigen, and is a rapid, inexpensive method enabling the presumptive,identification of positive enrichment cultures from a variety of foods, including ground beef, processed,meats, fruits and vegetables (9.1, 9.2). The polymyxin-ELISA utilizes a 96-well microtiter plate format,suitable for processing large numbers of samples.,3. DEFINITION OF TERMS,See Appendix A of Volume 3.,4. COLLECTION OF SAMPLES,See Appendix B of Volume 3.,5. MATERIALS AND EQUIPMENT,MFLP-92,- 2 - June 2005,Note: The laboratory supervisor must ensure that completion of the analysis described in this method is done,in accordance with the International Standard referred to as “ISO/IEC 17025:1999 (or latest version):,General requirements for the competence of testing and calibration laboratories”.,The following chemicals, materials and equipment are required to operate the method, and are,commercially available. See also section 8 of this method for the preparation of individual media. This is,in addition to the materials and equipment specified in the Health Canada Escherichia coli O157:H7,culture technique, MFLP-80 (9.3).,Note: If the analyst uses any variations of the media listed here (either product that is commercially available,or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure,equivalency.,1) Sodium phosphate, dibasic anhydrous,2) Sodium phosphate, monobasic monohydrate,3) Sodium chloride,4) Sulfuric acid,5) Polymyxin B sulfate,6) Sodium cholate,7) Tween 20,8) Peroxidase-labelled affinity purified antibody to E. coli O157:H7 (No. 04-95-90, Kirkegaard,& Perry Laboratories, Inc., Gaithersburg, MD),9) 3,3', 5,5' - tetramethylbenzidine (TMB) Microwell peroxidase substrate 1-component (No.,50-76-05, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD).,10) Phosphate Buffered Saline (PBS),11) PBS with Tween 20 (PBST),12) PBST with blocking protein (PBST-B),13) Polymyxin coating solution (PCS),14) Cholate extraction buffer (CEB),15) Stop solution,16) Non-fat dry milk blotting grade blocker (No. 170-6404, Bio-Rad Laboratories, Hercules,CA).,17) Nunc-Immuno MaxiSorp microtiter plates (VWR International, Mississauga, Ontario), flat,bottom,18) Microfuge tubes with caps (1.5 mL-capacity).,19) Microtiter plate reader capable of measuring absorption at 450 nm (e.g., Quant Microplate,Reader, Bio-Tek Instruments, Inc., Winooski, VT).,20) Microtiter plate washer (e.g., Wellwash AC plate washer, Thermo Labsystems, Helsinki,Finland),MFLP-92,- 3 - June 2005,21) Incubator capable of holding 37oC,22) Block heater capable of accommodating 1.5 mL-capacity microfuge tubes and attaining a,temperature of 100oC, or alternatively, boiling water bath,6. PROCEDURE,6.1 Handling of samples,Food Samples are handled and subjected to enrichment in modified trypticase soy broth with,novobiocin according to the Health Canada Escherichia coli O157:H7 culture technique, MFLP-80,(9.3). Enrichment broth cultures are then sampled and subjected to the polymyxin-ELISA,procedure as described below. A positive control consisting of a broth culture of a well,characterized Escherichia ……
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