IAFP 2009 Program Book and Abstract Book - AB104

Purpose: Based on this report, the [TA10] Pathogenic Bacterial Multiplex PCR Detection System was developed as a useful tool for detecting pathogens in food industries. Methods: The enrichment medium, DNA extraction kit, and multiplex PCR kit were supplied by a Japanese commercial company. The evaluation study was performed in 3 food company laboratories and 2 independent food research institutes. A total of 7 types of food matrices [55 kinds (cheese, apple juice, meat, fresh vegetable, fruits etc…)] of spiked food samples (inoculation level 5 CFU per 25 g) were analyzed with the detection kits, and the results were compared with the results of conventional culture methods in each collaborating laboratory. Results: The enrichment broth [TA10] was found suitable for enabling all pathogenic bacteria tested to exceed the lowest PCR detection limit (103 CFU/ml) within 20 h of incubation at 35°C, and the DNA extraction kit [TA10] in combination with the multiplex PCR detection kit [TA10] provides good recovery and amplification of target DNAs. The detection limit was in agreement (5 CFU per 25 g) within the laboratories, while 7 different types of spiked food samples were tested in three food company laboratories. Significance: [TA10] Pathogenic Bacterial Multiplex PCR Detection System (included pre-enrichment broth, DNA extraction kit, and multiplex PCR kit) can provide tools for a simple and highly sensitive detection method for pathogens in various food samples. No significant differences in detection sensitivity were seen among the evaluation studies performed in 3 food company laboratories. Therefore, this commercial kit could be a useful tool to help ensure of safety of food for consumers. P2-03 A Versatile Internal Control for DNA and RNA Real-time PCR Assays DEANNE M. DEER, Narjol Gonzalez-Escalona, Yi Chen and Keith A. Lampel FDA-CFSAN, College Park, MD, USA Introduction: Real-time polymerase chain reaction (qPCR) is a common technique for rapid detection and identification of pathogens in food and clinical samples. However, because of its sensitivity to inhibitors present in the samples analyzed, there is potential for false-negative results, with consequent assay detection failure. Purpose: The aim of this study was to develop a TaqMan-based internal control system (IC) that could be used as DNA in qPCR or as RNA in reverse transcription qPCR (qRT-PCR) to indicate the presence of assay inhibition and to multiplex the IC with existing bacterial detection methods. Methods: The internal control (myIC) was designed to be genetically dissimilar to all known organisms and created in a plasmid format with an upstream T7 promoter to generate RNA template as well as DNA. Forward and reverse primers were designed along with a Cy5-labeled TaqMan probe to use in conjunction with the unique template to create a complete internal control set. The myIC system was first tested independently in both DNA and RNA assays and then added to existing qPCR and qRT-PCR bacterial detection methods as an internal control. The multiplexed detection methods were tested against both pure cultures and artificially contaminated produce commodities. Results: The myIC system was shown to be an effective DNA-based internal control as part of a 4-plex qPCR method for the detection of Shigella spp. (pure culture and produce samples) and in a duplex qPCR method for the detection of Listeria monocytogenes. It was also a successful RNA-based internal control in the duplex qRT-PCR method of Salmonella spp. detection from jalapeno pepper samples. Significance: Because of the versatility of this internal control, the myIC system can supplement many new or existing qPCR and qRT-PCR pathogen detection methods, greatly reducing the number of false negatives and allowing fast determination of inhibitor presence in various matrices to be tested. While we have utilized myIC only in bacterial assays, its RNA capability makes it a potentially valuable internal control in viral detection as well. P2-04 Comparison of 3M™ Petrifilm™ Aerobic Count Plate Results for Raw and Processed Meat Samples after 24 Versus 48-h Incubation Roseane Machado, CRISTINA F. ABREU, Renata Lima and Adriana Tassinari 3M Brazil, Microbiology, Sumaré, Brazil Introduction: The 3M™ Petrifilm™ Aerobic Count (AC) Plate was developed for total aerobic bacteria enumeration in food and is an AOAC INTERNATIONAL Official Method of Analysis (OMA # 990.12). Purpose: The aim of the study was to compare AC Plate results following an incubation time of 24 h ± 2 h to results obtained by following the official method 48 h ± 3 h incubation time, for raw and processed meat samples (poultry, pig and beef ). Methods: 1024 meat samples were analyzed by five different meat processors, following the same protocol: 10 g of sample + 90 mL of Buffered Peptone Water to prepare the first dilution, followed by 1:10 serial dilutions. 1 mL of each dilution was plated onto AC Plates and incubated first at 35°C ± 1°C for 24 h ± 2 h. Colonies were counted and the results were recorded. These same plates were then incubated for an additional 24 h ± 2 h, and colonies enumerated. Results at 24 h ± 2 h and 48 h ± 3 h were reported as colony-forming units (CFUs)/g of meat. Results: 1,023 results were statistically analyzed, and results from one representative dilution of each sample were transformed to log base. Regression Analysis was R-Sq = 92.8% and Pearson correlation of log 24 h and log 48 h was 0.963. Significance: The statistical analysis indicates a strong linear correlation between 24 and 48 h results for AC Plates. The results suggest that naturally contaminated meat samples with aerobic mesophilic flora incubated on AC Plates following a 24 h ± 2 h incubation time may be a valid alternative to the same procedre with 48 h incubation, offering a reduction of 24 h in the incubation time of the AOAC OMA 990.12. Poster 104 — Abstract Book

IAFP 2009 Program Book and Abstract Book

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