Laboratory Procedure MFLP-30
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Health Products and Food Branch
This method is applicable to the detection of E. coli O157:H7 in raw ground beef and fruit juice to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act.
The BAX® system is a quick laboratory method for accurately detecting E. coli O157:H7 in raw ground beef and fruit juice. The BAX® system for E. coli O157:H7 detects the serotype in raw ground beef after 8 hours using the BAX® system media or 14 hours in modified E. coli Broth + 20 mg/L novobiocin, while the organism can be detected in fruit juice after 20 hours in modified E. coli broth + 20 mg/L novobiocin (mEC+n).
The system for E. coli O157:H7 is a convenient yes/no screening tool that uses Polymerase Chain Reaction (PCR) technology for reliable results 12-24 hours after sampling. The procedure takes about four hours after enrichment and involves about one hour of user time. BAX systems are designed for use by qualified lab personnel who follow standard microbiology practices.
The BAX® system for E. coli O157:H7 targets a specific fragment of bacterial DNA. This DNA fragment is stable, unaffected by the growth environment and is unique to E. coli O157:H7. PCR technology enables the BAX® system to provide rapid and specific DNA amplification with results that are available about four hours after enrichment.
PCR is a powerful means for quickly providing millions of copies of a specific DNA fragment. In a typical application, sample DNA is combined with polymerase, nucleotides and primers that are specific for a given nucleotide sequence. This mixture then undergoes a series of timed heating and cooling cycles. Heating denatures or separates the DNA into single strands, then as the mixture cools, primers recognize and anneal to the target DNA sequence. DNA polymerase then uses nucleotides to extend the primers, thereby creating two copies of the target DNA fragment. Repeated cycles of denaturing, annealing, and extending produce exponential increases in the number of target DNA fragments within a matter of hours. If the target sequence is not present, no detectable amplification takes place.
The BAX® system simplifies this process by combining the primers, polymerase, nucleotides and positive control into a single sample tablet that is already packaged inside the PCR tubes. Additionally, the automated fluorescent detection allows for closed-tube testing, virtually eliminating the potential for carry-over contamination with amplified DNA.
See Appendix A of Volume 3
See Appendix B of Volume 3
1) Supplied with kit - (no.17710611; sufficient for 96 tests; DuPont Qualicon, phone: 800-863-6842, fax: 302-695-5301)
2) Enrichment broth
3) Materials (provided with start-up package)
Lysis tubes with caps and racks
Tips for pipettes
Powder-free nitrile gloves
4) BAX® system with automated detection User Guide
7.1 Collect and enrich samples
Raw beef samples can be prepared with either modified EC broth with novobiocin (for 14-24 hour enrichment) or BAX® system media for E. coli O157:H7 (for 8-24 hour enrichment), depending on the needs and work flow of your lab. Fruit juices use modified EC broth with novobiocin for 14-24 hour enrichment.
7.1.1 Beef - Modified E. coli broth enrichment: Prepare samples at a 1:10 dilution in modified EC broth + 20 mg/L novobiocin. Incubate the enrichments at 35-37ºC for 14-24 hours.
7.1.2 Beef - BAX® system media enrichment: Prepare the required volume of BAX® system media for E. coli O157:H7 according to instructions on the container label. Add raw beef to pre-warmed (41-42ºC) broth at a 1:10 dilution. Incubate at 41-42ºC for 8-24 hours.
7.1.3 Fruit juices: Prepare samples at a 1:10 dilution in modified EC broth + 20 mg/L novobiocin. Incubate the enrichments at 35-37ºC for 14-24 hours.
7.2 Prepare equipment
7.2.1 Turn on heating blocks, and check that temperatures are set to 37ºC and 95ºC.
7.2.2 Initialize cycler/detector as outlined in BAX® system with automated detection User Guide, pages II-4 to II-14.
7.3 Process the samples
7.3.1 Lyse samples
184.108.40.206 Prepare the lysis tubes and transfer samples
220.127.116.11 Arrange the required number of lysis tubes (one for each sample and one for the "blank") in the rack.
18.104.22.168 Prepare lysis reagent by pipetting 150 μL of protease into one bottle of the lysis buffer.
22.214.171.124 Add 200 μL of lysis reagent to each lysis tube.
126.96.36.199 Transfer 5 μL of enriched sample to the corresponding lysis tube.
188.8.131.52 Secure the caps after all transfers have been completed.
7.3.2 Perform lysis
184.108.40.206 Heat the tubes at 37ºC for 20 minutes, then at 95ºC for 10 minutes.
220.127.116.11 Place the lysis tubes in cooling block for at least 5 minutes.
7.3.3 Prepare the PCR tubes and transfer lysate
18.104.22.168 Place PCR tube holder into PCR cooling block.
22.214.171.124 Place one PCR tube per sample in the holder.
126.96.36.199 Remove and discard lid from one strip of tubes at a time. Using multichannel pipettor, transfer 50 μL of each lysed sample into a corresponding PCR tube. Cover tubes with a new optical cap strip and secure tightly.
188.8.131.52 Repeat for all samples.
184.108.40.206 Take the entire cooling block to the cycler/detector. Samples should remain in the cooling block until the cycler/detector is ready for loading, but no more than 30 minutes after tablet hydration.
7.4 Amplify the DNA
Follow the screen prompts in the PCR Wizard to load your samples, run the program and unload your samples. (See BAX® system with automated detection User Guide for reference.)
7.5 Review displayed results
7.6 Confirm positive results
Positive results must be confirmed culturally as described in MFLP-80. Prepare appropriate dilutions from the enrichments. Spread plate to specified selective agars and confirm biochemically and serologically.