MFLP-97 The Alert Test Kit for Detecting Salmonella
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897FFEC07F0543CDBDD551871E07036F |
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0.02 |
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5 |
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日期： |
2024-7-30 |
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Published on the Food Directorate’s (Health Canada's) website at http://www.hc-sc.gc.ca/food-aliment.,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-97,February 2001,THE ALERT TEST KIT FOR DETECTING SALMONELLA,1. APPLICATION,The method is applicable to the detection of Salmonella from feeds, raw foods and other samples to,determine compliance with Sections 4 and 7 of the Food and Drugs Act. Where stipulated, the Official,Methods and HPB Method should be used.,2. DESCRIPTION,The method has been shown to produce satisfactory results with artificially contaminated foods and,naturally contaminated poultry rinse, raw ground turkey and rendered poultry meal in AOAC studies,(8.1). This method can be used successfully for the detection of Salmonella in other foods and food,ingredients.,3. PRINCIPLE,Alert for Salmonella is a sandwich ELISA utilizing both monoclonal and polyclonal antibodies made,against a Salmonella antigen. Following selective enrichment in tetrathionate broth and rappaportvasiliadis,broth and post enrichment in M broth, test cultures are heated to release the antigen and then,added to a microwell containing the bound polyclonal antibodies. During this first incubation, the antigen,is captured by the bound antibodies. After a wash step, peroxidase-conjugated anti-Salmonella,monoclonal antibody is added and allowed to react with the captured antigen. After a second wash,substrate is added which reacts with the peroxidase complex and develops blue color. The more blue,color is indicative of the more Salmonella antigen present in the sample culture. A stop solution is,added and the microwells are read on an EIA reader at 450 nm.,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,The media listed under No. 2 to 11 are commercially available and are to be prepared and sterilized,according to the manufacturer’s instructions.,1) Alert Test Kit (Neogen Corporation, phone: (517) 372-9200, Fax: (517) 372-0108),2) Lactose broth,3) Tryptic soy broth,- 2 - MFLP-97,February 2001,4) Universal Preenrichment broth,5) Brilliant green dye,6) Rappaport-Vasiliadis broth (RV),7) Tetrathionate broth (TT),8) M broth,9) Xylose lysine desoxycholate agar (XLD),10) Hektoen agar (HE),11) Bismuth sulfite agar (BS),12) Brilliant green sulpha agar (BGS),13) Incubator capable of maintaining 35EC,14) Incubator 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 35EC is recommended in the text,of the method the incubator may be at 35 +/-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.,15) Control cultures (ATCC or equivalent),16) 1N HCL and 1N NaOH,17) pH meter or paper,18) Stomacher blender or equivalent,19) Water-bath (100° C) or equivalent system,20) EIA/microwell reader at 450 nm,21) Biochemicals and antisera listed in MFHPB-20,7. 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 Thaw frozen samples in a refrigerator, or under time and temperature conditions which,prevent microbial growth or death.,71.2 Analyze sample units as soon as possible after their receipt in the laboratory.,- 3 - MFLP-97,February 2001,7.2 Preparation for Analysis,7.2.1 Have ready lactose broth, tryptic soy broth or universal preenrichment broth (see Table 1).,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 Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL (the analytical unit),into 225 mL of the required preenrichment broth, as indicated in Table 1.,7.3.3 Adjust pH of the mixture, if necessary, to 6.8 ± 0.2 with 1 N NaOH or 1 N HCl.,7.3.4 Incubate pre-enrichment mixture 18-24 h at 36EC.,7.4 Selective Enrichm……
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