MFLP-24 Identification of Escherichia coli O157 by the Warnex Real Time Polymerase Chain Reaction System
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D1BFDE82BDC74B8287BF7DDF08874229 |
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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-24,May 2005,HEALTH PRODUCTS AND FOOD BRANCH,OTTAWA,IDENTIFICATION OF ESCHERICHIA COLI O157 BY THE WARNEXTM,REAL TIME POLYMERASE CHAIN REACTION SYSTEM,Stephen Shaw and Jessica Bosley,Research and Development Section, Ottawa Laboratory Carling (OLC),Canadian Food Inspection Agency,Ottawa, Ontario, K1A 0C6,E-mail: sshaw@inspection.gc.ca,E-mail: bosleyj@inspection.gc.ca,1. APPLICATION,The method is applicable to the rapid identification of Escherichia coli O157 strains (E. coli O157) isolated,from foods and food ingredients using the standard E. coli O157 culture technique, MFLP-80 (6.1), other,HPFB methodologies and other non-compendium methodologies to detect E. coli O157. It can be applied,to the presumptive identification of E. coli O157 from enrichment broth culture. When product-based,compliance action is anticipated, and where stipulated, HPFB methodology should be used exclusively or,for further confirmation of real time polymerase chain reaction (PCR) positive colonies.,This revised method replaces MFLP-24, dated December 2003.,2. PRINCIPLE,Following the sample enrichment procedure for food and food ingredients, the broth is subjected to a real,time PCR procedure which amplifies a unique specific fragment of DNA sequence to the E. coli O157,gene. The oligonucleotide primers used in the fluorescent PCR system are highly specific for E. coli,O157, and do not amplify DNA present in other non-E. coli O157 organisms under the reaction conditions.,The resulting amplified DNA fragment has a specific molecular size, defined by the selected primers, and,is readily identified by the E. coli O157 specific fluorescent probe. The entire procedure, after the,enrichment, identifies presumptive positive samples within 3 hours, and can replace the usual screening,tests thus providing considerable savings on time, labour and cost of the analysis. The real time PCR,technique has proven to be a specific and sensitive method for the presumptive identification of E. coli,O157 from a variety of samples.,The WarnexTM real time PCR rapid pathogen detection system for E. coli O157 has been AOAC-RI,validated and was granted “Performance Tested Method” status in 2004, Certificate No. 010408.,* WarnexTM is a trademark of Warnex Diagnostics Inc., 3885 Industriel Blvd., Laval (Quebec), Canada,H7L 4S3.,Ph: (450) 663-6724, Fax: (450) 669-2784, website: www.warnex.ca,3. SPECIALIZED PRIMERS, REAGENTS, BUFFERS, MATERIALS AND EQUIPMENT,MFLP-24,- 2 - May 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”.,3.1. MATERIALS AND EQUIPMENT,3.1.1 Materials and special equipment provided,Extraction buffer (EX-1),Extraction reagent, lyophilized (EX-2),Extraction plates,Plate seals,Detection buffer (DT-1),Detection reagent, lyophilized (DT-2),Detection plates (containing pre-dispensed PCR reagents and primers),PCR optical-grade plastic caps,3.1.2 Additional materials and special equipment required,Warnex-validated Real-Time PCR thermocycler *,* Thermocycler specifications:,- 96 well low profile microplate or 96 x 0.2 mL low profile strip tube sample capacity.,- ability to excite fluorophores with a peak excitation range of 485 to 520 nm,- ability to detect fluorophores with a peak emission range of 500 to 600 nm.,Vortex (with platform head adaptors and replacement inserts),Centrifuge (with rotor and plate adaptor),Stomacher,Stomacher filter bags,Pipettors to cover range of volumes (0.5-1000 :L) with sterile plugged pipette tips.,3.2 PCR PRIMERS, TEMPERATURE CYCLING PROGRAM, BUFFERS AND REAGENTS,3.2.1 PCR Primers,The oligonucleotide primers and molecular beacon probes are supplied in the kit and their,specificity for E. coli O157 has been verified (6.2).,3.2.2 Temperature cycling programs,The temperature cycling programs for the PCR include an automated 15-minute,extraction cycle, resulting in the lysis of the bacterial cells, followed by an automated PCR,detection process. The thermal cycler program for the detection (amplification) process is,preset for the following sequence of cycling parameters:,Amplification:,1 cycle of:,Hot start, 15 mins, 95°C,40 cycles of:,Denaturation, 15 secs, 94°C,Annealing, 15 secs, 55°C,Plate read,Extension, 15 secs, 72°C,MFLP-24,- 3 - May 2005,3.2.3 Buffers and Reagents,All b……
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