AOAC RI ERP Final Action Recommendations eBook

Dupont BAX Salmonella PTM Report Modification Approved 2012 / PTM Certification No. 081201 For Expert Review Panel Use Only Do Not Distribute

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reducing the potential for contamination with one or more molecules of amplified PCR product in

future tests.

Fluorescent real time detection [1] - This automated BAX® System method uses fluorescent

detection to analyze PCR product. One PCR primer for each target (one Salmonella- specific target

and an internal control) contains a fluorescent dye (two different dyes, one for each target) as a

constituent of the primer as well as a quencher (the uni-molecular combination of a primer,

fluorescent dye and quencher constitute a Scorpion™ probe). When not incorporated into a PCR

product, the Scorpion™ probe has a hair-pin loop structure which keeps the dye and quencher in

close proximity. When incorporated into a PCR product, the dye and quencher are spatially

separated due to an internal hybridization, which causes an increase in emission signal.

The BAX® System measures the magnitude and characteristics of fluorescent signal change

during each cycle of PCR. The data points generated are analyzed using an algorithm which

evaluates the increase in signal intensity, the slope of the rise relative to increase in signal, and the

noisiness of the baseline for each sample. If the amplification plot represents the sigmoid shape

characteristic of the targeted PCR response, the software assigns a positive result to that sample. If

there is no evidence of the exponential amplification event, the software assigns a negative result to

that sample. In cases where there is anomalous data, such as an unexpected degree of noise or a

large rise in fluorescence which cannot be modeled as an exponential amplification, the software

assigns an indeterminate result.

AOAC INTERNATIONAL 6. GENERAL INFORMATION Salmonella is a foodborne pathogen that traditionally has been screened for by culture

methods, such as those of the Bacteriological Analytical Manual (BAM) of the United States

Food and Drug Administration [2], the Health Canada Compendium of Analytical Methods [3],

and the Microbiological Laboratory Guidebook (MLG) of the United States Department of

Agriculture Food Safety and Inspection Service (USDA-FSIS) [4]. These culture methods each

require at least three days for a negative result and often many more days if there are potential