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DuPont™ BAX
®
System Real-Time PCR Assay for
Salmonella
: Collaborative Study
DuPont Nutrition & Health
Page 4
To create 325 g test portions, 25 g of this spiked bulk quantity was added to 300 g uncontaminated
1
frankfurter and mixed to combine.
2
To inoculate orange juice, a pure colony of
Salmonella
Hadar was transferred from SBA into BHI broth
3
containing 1% glucose and incubated at 35°C for 18-24 hours. This stress protocol resulted in a percent
4
injury of approximately 60% (as determined by plating onto selective Xylose Lysine Desoxycholate agar
5
and non-selective Tryptic Soy agar). The inoculum was added drop-wise to a bulk quantity of orange
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juice to reach the desired contamination level, then mixed to achieve equal distribution of the inoculum
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throughout. This spiked bulk quantity was divided into 25 mL test portions for analysis.
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All test portions were randomized and blind-coded by the organizing laboratory, then shipped overnight
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to each collaborating laboratory and maintained at 2-8°C until they were analyzed. The total hold time
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of samples was 48 hours for frankfurters and 96 hours for orange juice, including shipment time to each
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participating laboratory. For each sample type, each collaborator received twelve replicate test portions
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per sample per inoculation level with the candidate method and the reference method. On the first day
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of test sample analysis, a 5-tube, 3-level Most Probably Number (MPN) estimation of contamination
14
levels was conducted using the appropriate reference method. The Least Cost Formulations, Ltd. (LCF)
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MPN Calculator-Version 1.6 [5] was used to determine the MPN values and 95% confidence intervals.
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The MPN is reported in the results tables below as MPN/test portion with 95% confidence intervals.
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AOAC Official Method ####
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AOAC Research Institute Performance Tested Method 081201 2012
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AOAC International First Action Official Methods of Analysis 2013
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DuPont™ BAX® System Real-Time PCR Assay for
Salmonella
- Method for Detection of
Salmonella
in
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Foods and Selected Environmental Surfaces
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(Applicable to the detection of
Salmonella
in a variety of foods, including raw ground beef , ground
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beef with soy , beef trim, frankfurters , shrimp, ground turkey, chicken wings, poultry rinse, whole
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powdered (dried) eggs, shell eggs, fresh bagged lettuce, frozen peas, orange juice [pasteurized], cream
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cheese, nonfat dry milk, ice cream , peanut butter, cocoa, white pepper, milk-based infant formula,
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and dry pet food, and on stainless steel, ceramic tile, and plastic surfaces)
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Safety Precautions
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Kits – The reagents used in the BAX® System should pose no hazards when used as directed. Dispose of
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lysate, PCR mixture and other waste according to your site practices.
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Cycler/detector – Only qualified laboratory personnel should operate the cycler/detector. Do not
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attempt to repair the instrument. Live power may still be available inside the unit even when a fuse has
32
blown or been removed. Refer to the BAX® System User Guide for maintenance procedures when
33
cleaning the unit or changing a fuse. The heating block can become hot enough during normal operation
34
to cause burns or cause liquids to boil. Wear safety glasses or other eye protection at all times during
35
operation.
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FINAL (Version 4)