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1.0
Introduction
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The purpose of this collaborative study is to determine the performance of the candidate method
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among collaborators for the detection of
Salmonella
in a variety of foods. Estimates of repeatability,
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reproducibility, probability of detection (POD), and Relative Limit of Detection (RLOD) will be evaluated.
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Collaborators will analyze 2 matrices at 3 contamination levels comparing the performance of the
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candidate method to appropriate reference culture methods. The BAX® System Real-Time PCR Assay for
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Salmonella
is the candidate method.
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1.1
BAX® System Real-Time PCR Assay for
Salmonella
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The BAX® System Real-Time PCR Assay for
Salmonella
uses the Polymerase Chain Reaction (PCR)
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to amplify a specific fragment of bacterial DNA, which is stable and unaffected by growth
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environment. The fragment is a genetic sequence that is unique to the genus
Salmonella
, thus
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providing a highly reliable indicator that the organism is present. The BAX® System simplifies the
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PCR process by combining the requisite primers, polymerase and nucleotides into a stable, dry,
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manufactured tablet already packaged inside the PCR tubes. After amplification, these tubes
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remain sealed for the detection phase, thus significantly reducing the potential for
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contamination with one or more molecules of amplified PCR product.
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This automated BAX® System method uses fluorescent detection to analyze PCR product. One
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PCR primer for each target (one
Salmonella-
specific target and an internal control) contains a
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fluorescent dye (two different dyes, one for each target) as a constituent of the primer as well as
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a quencher (the uni-molecular combination of a primer, fluorescent dye and quencher
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constitute a Scorpion™ Probe). When incorporated into a PCR product, the dye and quencher
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are spatially separated, which causes an increase in emission signal. The BAX® System measures
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the magnitude and characteristics of fluorescent signal change. An analysis by the BAX® System
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software algorithm then evaluates that data to determine a positive or negative result which is
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displayed as described below.
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1.2
Summary of the
Performance Tested Method
/Pre-collaborative study
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The BAX® System Real-Time PCR Assay for
Salmonella
was certified by the AOAC Research
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Institute in August, 2012 and designated
Performance Tested Method
SM
(PTM) #081201. No
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significant differences were reported for detection of
Salmonella
in the matrices tested when
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comparing the BAX method results to the following standard reference culture procedures; U.S.
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Department of Agriculture-Food Safety and Inspection Service
Microbiological Laboratory
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Guidebook
(USDA-FSIS MLG) Chapter 4.05 (1), U.S. Food and Drug Administration
Bacteriological
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Analytical Manual
(FDA BAM) Chapter 5 (2), and Health Canada
Compendium of Analytical
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Methods
(HC CAM) MFHPB-20 (3). The matrices validated in the PTM study, along with the
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appropriate reference method used for comparison, include the following: raw ground beef (25
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Collaborative Study Approved Protocol
Expert Review Panel Use Only