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P2-20 Evaluation of FDA Developed Real-time Quantitative PCR (qPCR) Using Two Different PCR Master Mixes for the Detection of Salmonella in Leafy Greens Using Five Different Pre-enrichment Media

Monday, July 27, 2015
Exhibit Hall (Oregon Convention Center)
Hua Wang , U.S. Food and Drug Administration , College Park , MD
Andrew Jacobson , U.S. Food and Drug Adminstration , College Park , MD
Anna Laasri , U.S. Food and Drug Administration-CFSAN , College Park , MD
Aparna Tatavarthy , U.S. Food and Drug Administration , College Park , MD
Chorng-Ming Cheng , U.S. Food and Drug Administration , Irvine , CA
Vikas Gill , U.S. Food and Drug Administration , College Park , MD
Thomas Hammack , U.S. Food and Drug Administration , College Park , MD
Introduction: Salmonella outbreaks traced to leafy greens show the need for rapid and reliable detection methods for fresh produce. The Bacteriological Analytical Manual (BAM) culture method for Salmonella uses lactose broth (LB) for pre-enrichment, which has not reliably supported qPCR assays as shown in our former studies.

Purpose: The performances of FDA qPCR assay to detect Salmonella in lettuce, parsley, cabbage, and spinach was evaluated using two different PCR master mixes (VeriQuestTM and illustraTM PCR beads) and ABI 7500Fast real-time PCR detection system in LB and four other pre-enrichment media: buffered peptone water (BPW), modified BPW (mBPW), Universal Pre-enrichment broth (UPB), and BAX®MP media.

Methods: Produce, equivalent to at least 100 test portions, was inoculated with a single Salmonella serovar and stored at 2 - 8ºC for 3 d prior to analysis. On the day of analysis, twenty test portions (25 g) from the bulk inoculated produce were prepared for each of the five pre-enrichment media, and then 225 ml pre-enrichment media was added to respective test portions. The BAM culture method was followed thereafter. qPCR was performed from 24 h pre-enriched cultures.

Results: No significant differences (P > 0.05) were found among the five media for leafy greens by culture results, but LB was the least effective broth. qPCR using both PCR master mixes produced significantly (P < 0.05) higher false negatives in 24 h pre-enriched LB than the other four media. The VeriQuest mix provided more sensitivity than PCR beads and culture methods in all five media.

Significance: This study addressed a need to improve current BAM Salmonella culture method for the detection of Salmonella from leafy greens when using real-time PCR as screening method.

Back to: Poster Session 2 – Laboratory and Detection Methods; Meat, Poultry and Eggs; Dairy and Beverages; Food Law and Regulation; General Microbiology; Modeling and Risk Assessment
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