MFLP-14 The Genequence Listeria Monocytogenes Assay for the Detection of Listeria monocytogenes in a Variety of Foods
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6683EEF8E946404FB893F8C31C150F00 |
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文件大小(MB)： |
0.06 |
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页数： |
7 |
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日期： |
2012-3-2 |
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Published on the Food Directorate’s (Health Canada's) website at:,http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php,Government of Canada Gouvernement du Canada,Laboratory Procedure,MFLP-14,February 2011,Health Products and Food Branch,Ottawa,The Genequence Listeria monocytogenes assay for the detection of,Listeria monocytogenes in foods,Don Warburton,Evaluation Division,Bureau of Microbial Hazards, Food Directorate,Postal Locator: 2204E,HPFB, Ottawa, Ontario, K1A 0K9,E-mail: micro_methods_committee@hc-sc.gc.ca,1. Application,This method is applicable to the rapid detection of Listeria monocytogenes to determine compliance with the,requirements of Sections 4 and 7 of the Food and Drugs Act. Positive results must be confirmed with a,cultural method. This method has been validated for use in raw meat. Insufficient validation data have been,received for hot dogs, deli ham, deli turkey, ice cream, pasteurized milk, brie cheese, parmesan cheese,smoked salmon, cooked crab meat, lettuce, frozen peas, soy flour and environmental samples. This revised,method replaces MFLP-14, dated November 2005.,NOTE: While this method is only approved for certain food products, as listed above, it is assumed that this,method could be used with other commodities. To ensure the method is fit for purpose for,commodities outside the application, it is imperative that other commodities be properly validated,following the criteria in the Compendium of Analytical Methods. It is requested that these validation,data be forwarded to the Microbiological Methods Committee so the Application Section can be,expanded to include these new foods if the data fulfill MMC requirements (refer to Development of,Methods in Volume 1 of the Compendium of Analytical Methods).,2. Description,This test is intended to qualitatively detect Listeria monocytogenes in foods, and should be used by,personnel with appropriate laboratory training in microbiology. This DNA hybridization test employs L.,monocytogenes-specific DNA probes directly labeled with horseradish peroxidase, and a colorimetric,endpoint to detect as little as one CFU of L. monocytogenes in a 25 g food sample when the specified,enrichment protocols are used.,MFLP-14,February 2011,Published on the Food Directorate’s (Health Canada's) website at:,http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php,- 2 -,3. Principle,The GeneQuence DNA hybridization test employs Listeria monocytogenes specific DNA probes labeled with,horseradish peroxidase and a colorimetric endpoint for the detection of L. monocytogenes in food samples,following broth culture enrichment. After sample pre-enrichment and a period of selective enrichment, target,cells are lysed enzymatically at 371C and L. monocytogenes B specific oligonucleotide probes are added for,a 60 minute hybridization incubation at 45°C. If L. monocytogenes ribosomal RNA (rRNA) is present in the,test sample, detector probe directly labeled with horseradish peroxidase (HRP) and polydeoxyadenylic acid,(poly dA)-tailed capture probe hybridize to the target organism rRNA sequences. Concurrently, base pairing,between the poly dA-tailed capture probe and polydeoxythymidylic acid (poly dT) coated polystyrene,microwells facilitates solid phase capture of the probe-target hybrid molecules. Unbound probe is removed,by washing and substrate-chromogen is added to each well. The reaction of the HRP with substratechromogen,produces a blue color. The reaction is stopped with the addition of sulfuric acid, which changes,the color of the substrate from blue to yellow. A microwell plate or microwell strip reader (A450) measures,absorbance; absorbance in excess of the threshold value indicates the presumptive presence of L.,monocytogenes in the test sample.,4. Definition of Terms,See Appendix A of Volume 3.,5. Collection of samples,See Appendix B of Volume 3.,6. Materials and special equipment,NOTE: The Laboratory Supervisor must ensure that the analysis described in this method is carried out,in accordance with the International Standard referred to as AISO/IEC 17025:1999 (or latest,version): General Requirements for the Competence of Testing and Calibration Laboratories.@,The media listed below are commercially available and are to be prepared and sterilized according to the,manufacturer=s instructions. See also Appendix G of Volume 3 for the formula of individual media.,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 for Laboratory Supervisor to ensure equivalency.,6.1 Supplied……
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