IAFP 2009 Program Book and Abstract Book - AB118

P2-38 DNA Aptamers with Binding Specificity for Campylobacter jejuni: Application to Pre-analytical Sample DSC Processing HARI PRAKASH DWIVEDI, Ronald D. Smiley and Lee-Ann Jaykus North Carolina State University, Raleigh, NC, USA Introduction: Antibodies are widely used for pathogen concentration and purification prior to detection. Alternative ligands, including nucleic acid aptamers, have the potential to provide binding specificity equivalent to antibodies but with advantages such as low cost, ease of manufacture, and improved stability. Purpose: To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni, and to apply these to pre-analytical sample processing prior to PCR-based detection. Methods: To identify candidate aptamers, a whole cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) method was applied to a combinatorial library of FAM-labeled single stranded (ss) DNA molecules. A total of ten SELEX rounds were applied, using C. jejuni strain A9a as the target. Periodically, binding was confirmed using flow cytometry and the aptamers demonstrating peak fluorescence were sorted. Selectivity of the aptamers was further enhanced using counter-SELEX methods. The enriched aptamers were cloned, sequenced and analyzed for binding characteristics. Results: Nine candidate FAM-labeled aptamer sequences with high binding specificity to C. jejuni were identified upon challenge with 108 to 109 C. jejuni (A9a) cells. Aptamer ONS-23 showed the highest binding affinity [dissociation constant (Kd value) of 292.8 ± 5.3 nM], with 47 ± 6% of cells fluorescent (bound) at a 1.48 µM aptamer concentration. Aptamer inclusivity was demonstrated by similarly high binding efficiencies (ranging from 25 to 36%) of ONS-23 for different C. jejuni strains including ATCC-33560, ATCC-33291, strain 2083, and strain A14a. Binding specificity was confirmed by challenging aptamer ONS-23 with other foodborne bacteria including Bacillus cereus strain T, E. coli O157: H7, and L. monocytogenes ATCC 19115, for which binding efficiencies never exceeded 5%. Significance: This project demonstrates that DNA aptamers can be selected using a whole cell-SELEX method, with resulting aptamers demonstrating high binding affinity and specificity. Aptamers are currently being evaluated for their ability to capture C. jejuni from complex sample matrices for subsequent detection by PCR. P2-39 Comparison of Real-time PCR and Conventional Culture Method for Detection of Campylobacter jejuni in Ground Beef and Vegetable Salad JEONG HWAN CHEON, Ji-Yeon Hyeon, Jae-Hoon Lee, Kwang-Young Song, Jong Seok Park, Seok Heo, Se Wook Oh and Kun Ho Seo Konkuk University, College of Veterinary Medicine, Seoul, Korea, South Introduction: Campylobacter jejuni (C. jejuni) is the leading cause of foodborne gastroenteritis worldwide. Rapid and effective methods have been developed, as the standard culture method requires up to 5 days and is not suitable for routine testing of large numbers of samples. Real-time PCR was evaluated for the timesaving and sensitive detection of C. jejuni in food samples using molecular beacon probes. Purpose: The purpose of this study was to compare the performance of real-time PCR and culture method for the detection of C. jejuni in ground beef and vegetable salad. Methods: Performances of two detection methods (real-time PCR and FDA/BAM reference method) were evaluated for recovery of C. jejuni in artificially contaminated food samples. Five hundred grams of samples (ground beef and vegetable salad) were artificially contaminated with various concentrations to generate partial positive, then divided into 20 samples (25g each). Samples were incubated in Bolton broth at 37°C (pre-enrichment for 4 h) and 42°C (enrichment for 44 h), then streaked onto the mCCD agar incubating under microaerobic conditions at 42°C for 48 h, followed by a confirmation test using API Campy. In parallel, real-time PCR was performed using primers and probes targeting the open reading frame (ORF) C sequence specific for C. jejuni after enrichment in Bolton broth. Results: In ground beef, real-time PCR detected the same number or more positives than the culture method, but there was no significant statistical difference (real-time PCR: 65/80, culture method: 59/80, P = 0.3440). In vegetable salad, in contrast, the number of positive samples by real-time PCR was much more than with the culture method. In addition, there was an extremely significant statistical difference (real-time PCR: 37/80, culture method: 1/80, P < 0.0001) between the two methods. Significance: Real-time PCR provides more reliable detection of C. jejuni than the conventional culture method with fewer steps, especially for foods with high numbers of background flora, like vegetable salad. Poster P2-40 Immunomagnetic Concentration and Real-time RT-PCR Detection of Norovirus SOPHIE ROY, Solange E. Ngazoa, Andre Darveau and Julie Jean Universite Laval, Institute of Nutraceuticals and Functional Foods, Quebec, QC, Canada Introduction: Norovirus (NoV) is the major agent of acute foodborne gastroenteritis in human worldwide. The concentration of viral particles found in food samples is very low but still sufficient to cause infections in humans and corresponds to the minimal infectious dose. Therefore, the concentration of viral particles prior to detection is necessary to achieve an accurate and sensitive detection of these viruses in clinical, food and environmental samples. Purpose: The objective of this study was to develop and evaluate the efficacy of an immunomagnetic concentration method combined to real-time RT-PCR for the sensitive and specific detection of NoV. Methods: Polyclonal antibodies have been produced, using a synthetic peptide of the viral capsid protein as an antigen. The antibodies obtained were purified and immobilized on magnetic beads by covalent link. The magnetic beads capturing viral particles were lysed and submitted to amplification by TaqMan RT-PCR. The concentration and detection approach has been applied to different spiked food samples. 118 — Abstract Book

IAFP 2009 Program Book and Abstract Book

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