MFLP-86 Identification of Presumptive Positive Verocytoxigenic Escherichia coli by the Polymerase Chain Reaction
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836F04FD2A0642BD83D71D40086B22DB |
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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-86,January 2003,IDENTIFICATION OF PRESUMPTIVE POSITIVE VEROCYTOTOXIGENIC,ESCHERICHIA COLI BY THE POLYMERASE CHAIN REACTION,Kim Ziebell,Laboratory for Foodborne Zoonoses,Health Canada,Guelph, Ontario N1G 3W4,E-mail: Kim_Ziebell@hc-sc.gc.ca,1. APPLICATION,The method is applicable to the rapid identification of Verocytotoxigenic Escherichia coli (VTEC) isolated from,foods and other samples using APHD Methodology (6.1), HPFB methodology (6.3) and other methodology to,detect Escherichia coli O157 and/or other VTEC. It can be applied to the definitive identification of presumptive,positive colonies isolated on MacConkey agar, and/or any suitable agar medium routinely used in the isolation,of this pathogen from enrichment cultures . When product-based compliance action is anticipated, and where,stipulated, HPFB Methodology should be used exclusively or for further confirmation of polymerase chain,reaction-positive colonies.,2. PRINCIPLE,Following the detection procedure, a portion of presumptive positive colonies plated on MacConkey Agar (Mac),is subjected to a polymerase chain reaction (PCR) procedure* which amplifies a specific DNA sequence of the,toxin gene. The priming oligonucleotides (primers) used in the PCR are highly specific for VTEC, and do not,amplify DNA present in any other non-VTEC organisms. The resulting amplified DNA fragment has a specific,molecular size, defined by the primers, and is readily identified by agarose gel electrophoresis. The entire,procedure identifies presumptive positive colonies within 5 h, and can replace the usual screening and,confirmation tests thus providing considerable savings on time, labour and cost of the analysis. The PCR,technique has proven to be a highly specific and sensitive method for the identification of VTEC from a variety,of samples.,*The polymerase chain reaction (PCR) process is covered by U.S. patents owned by Hoffman-LaRoche.,3. MATERIALS AND SPECIAL EQUIPMENT,1) Thermal cycler ( Model 9600, Perkin-Elmer Cetus) or equivalent.,2) Microwave oven or hot plate.,3) Submarine gel casting tray, buffer reservoir and power pack.,4) Shortwave UV light table (transilluminator) to visualize stained DNA in agarose gels.,MFLP-86,- 2 - January 2003,5) Photodocumentation system (optional, for photographic records), including polaroid camera (hand-held,or fixed),6) Pipettors: to cover range of volumes (0.5-1000/:L).,7) sterile Milli-que (MQ) water or equivalent (purified or distilled),4. PROCEDURE,Prepare and enrich samples according to APHD Methodology for isolation of Verocyto-toxigenic Escherichia,coli (Food Safety Procedures Manual, Chapter 6c) (6.1), HPFB methodology (MFLP-80) (6.3) or methodology,for detection of Escherichia coli O157 or other VTEC. At the stage where presumptive positive colonies are,observed on Mac Conkey agar or other selective agar, portions of the colonies are separately sampled and,subjected to the PCR procedure, which is carried out in accordance with the following instructions:,(Note: formulas and sources for all buffers and reagents used are given in Sections 7 and 8),4.1 Handling of samples,4.1.1 Sample units are handled and subjected to the enrichment, isolation and plating procedures,according to Agriculture and Agri-food Canada (6.1), HPFB methodology (6.3) or alternate,methodology. Presumptive positive VTEC colonies isolated on the selective agar plates (e.g.,Mac) are then sampled and subjected to the PCR method as indicated below. A positive,control consisting of a broth culture (Brain Heart Infusion) of a VTEC laboratory strain is also,processed along with each set of samples as well as a negative control consisting of a non-,VTEC organism.,4.2 Cell Iysis,4.2.1 Pick a small portion of a suspect colony (lightly touching the surface with a loop or needle is,sufficient) and suspend in 1 mL of BHI broth in a sterile 1.5 mL microfuge tube. For a negative,template control, leave one tube empty of culture (sterility control). Incubate at 35oC overnight.,4.2.2 Centrifuge cultures at 12,000 rpm for 1 min. and wash with 1 mL of FA buffer. Resuspend cell,pellet in 0.5 mL sterile Milli-que (MQ) water or equivalent.,4.2.3 Heat at 100/C for 10 minutes and immediately place on ice. Centrifuge the tube at 12,000 rpm,for 1 min.,4.3 PCR method,4.3.1 Add 5 :L of the cell Iysate or sterility control (see 4.2.1) to 20 :L of PCR reaction mixture in a,0.2 mL microfuge tube.,4.3.2 Cap the tubes and place in thermocycler and start program. After the PCR ……
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