IAFP 2009 Program Book and Abstract Book - AB109

Significance: The results suggest that a PCR system such as BAX is a faster and more sensitive method for detecting low levels of Salmonella in sprouts or spent irrigation water collected from sprouting of naturally contaminated seeds. P2-15 Comparison of Commercial RNA Extraction Kits for Preparation of DNA-free Total RNA from Salmonella Cells NARJOL GONZALEZ-ESCALONA and Benedicta Asamoah CFSAN/FDA, College Park, MD, USA Introduction: The isolation of DNA-free RNAs is a critical step for conducting RT-PCR assays. Nearly every RNA extraction procedure described previously has resulted in RNA samples being contaminated with genomic DNA. The consequence of this is, of course, a false positive result after RT-PCR. This is true, in particular, when real-time RT-PCR assays are employed for high sensitivity applications where false-positive results equate generally with a failure of the assay Purpose: The aim of this study was to compare some of the most commonly employed RNA extraction kits and a classic plasmid extraction procedure for determining which provides:(1) superior RNA recovery (e.g., the amount of RNA), (2) optimal purity (e.g., DNA contamination), (3) ease of deployment for high-throughput analysis and (4) the lowest price per reaction. Methods: RNA was extracted from exponential Salmonella SE5 cells using: RiboPure™-Bacteria Kit (Ambion), PureLink™ RNA Mini Kit (Invitrogen), UltraClean™ Microbial RNA Isolation Kit (MoBio), RNeasy Mini Kit (Qiagen), MasterPure™ RNA Purification Kit (EPICENTRE), and Plasmid DNA Isolation Reagent System (Carolina Biological). RNA concentration and initial purity was assessed through A260/280 ratio using Nanodrop. Further analysis for detecting genomic DNA contamination was performed by a quantitative reverse transcriptase real time PCR (RT-qPCR) targeting invA mRNA. Results: The purity of the RNA obtained with all of the kits, as assessed by the A260/280 ratio, was highly comparable at around 2.0. A higher total RNA concentration was obtained with the kit from EPICENTRE and with the plasmid extraction kit; however, DNA contamination (A280) was markedly high with these methods. Following in RNA recovery yield were the Invitrogen and Qiagen kits. RT-qPCR analysis of the samples before and after DNase I treatment showed that most kits performed similarly for invA mRNA detection save for the EPICENTRE and Mo Bio kits where DNA contamination was higher and interfered with the accuracy of the assay. Significance: This comparison showed that most of the kits analyzed are suitable for Salmonella RNA extraction. A higher RNA recovery was achieved with the EPICENTRE kit, but this kit also co-isolated high levels of genomic DNA which interfere with invA mRNA detection by RT-qPCR. Based on total RNA yield, levels of contaminant DNA, feasibility of highthroughput analysis, and price per reaction, the Qiagen and Invitrogen kits were shown to be the optimal choices for detection of invA mRNA by RT-qPCR. The use of these kits should greatly reduce the amount of false positives (i.e., DNA contamination) and will allow for a fast determination of invA mRNA in food samples. P2-16 Assessment of Rapid Pathogen Detection Kits for Salmonella on Melons for Test to Release Programs PAULA MARTINS DE FREITAS, Carol D’Lima and Trevor Suslow University of California, Plant Sciences, Davis, CA, USA Introduction: Occasional discrepancies between contract labs and regulatory labs in detection and recovery of Salmonella from melons, from a given lot, may be a reflection of non-homogeneous contamination, differences in general methods, and more recently, specific inherent differences between conventional and rapid test kit sensitivities. Purpose: To evaluate the specificity and sensitivity of rapid test kits considered for detection of Salmonella within ‘Test and Release’ protocols on cantaloupes and honeydews. Methods: Five platforms of combined recovery and detection were used: PATHATRIX, BAX®, Assurance GDS™, RapidChek® and Reveal®. Melons were surface inoculated with 36 or 100 CFU/melon of a three serovar cocktail of Salmonella enterica. After 5 days storage at 5°C, melons were peeled and 25 g, including the inoculated rind-spot, was subjected to the recommended protocol. In addition, over 200 non-inoculated melons were surveyed for Salmonella using the first three detection methods listed. Results: With 36 CFU/fruit, on cantaloupe, there were 100, 66 and 33 percent detection for BAX, PATHATRIX and RapidChek respectively. Reveal and GDS were negative for all inoculated melons. For honeydew, all test results were negative. At 100 CFU/fruit, 88% of cantaloupes were positive using BAX whereas 28% were positive, from the same enrichment culture, using GDS. All uninoculated controls were negative. Additional viability-confirmation controls demonstrated that honeydew had comparatively poor survival of Salmonella. In the expanded survey with uninoculated melons, 5 confirmed false positives were observed with BAX® on cantaloupes. Significance: Our outcome of approximately 2% ‘false positives’ would likely create management challenges if applied to Test to Release programs for cantaloupe, without protocol modification. With further melon-specific performance data, the sensitivity threshold for a positive detection outcome, in some regions, may be safely elevated to eliminate most false positive reactions. False negatives, especially with low levels of stressed cells, also remain an issue to resolve for routine compliance testing using rapid methods. P2-17 An Independent Evaluation of a New Method for the Detection of Salmonella in a Variety of Foods: The VIDAS ® Easy Salmonella Assay ERIN S. CROWLEY, Patrick Bird, Marianne Torontali, James Agin and David Goins Q Laboratories, Inc., Microbiology R&D, Cincinnati, OH, USA Introduction: Salmonella is one of the main causes of food poisoning; thus the rapid detection of this pathogen is a vital component of food safety. This new screening method detects Salmonella spp. in food and environmentals samples within 48 h. The alternative method utilizes a simple 2-step enrichment procedure for use with the enzyme linked immunofluorescent assay (ELFA). In the new enrichment protocol, a single selective proprietary broth (SX2) replaces the two broths Abstract Book — 109 Poster

IAFP 2009 Program Book and Abstract Book

Table of Contents for the Digital Edition of IAFP 2009 Program Book and Abstract Book