IAFP 2009 Program Book and Abstract Book - AB140

ratio, and stability against sedimentation in the absence of an applied magnetic field. These characteristics of nano-sized beads boost the binding efficiency by more than 20 to 30% and the available adsorptive areas by 100 to 1000 times. Purpose: The objective of the present study is to develop a specific, sensitive, and reproducible immunomagnetic separation concept, using magnetic nanoparticles with a diameter of 30 nm, for highly efficient separation of L. monocytogenes without any need for filtration or centrifugation steps. Methods: Magnetic nanobeads (30 nm) were functionalized with rabbit anti-Listeria monocytogenes antibodies via biotin-streptavidin bond and then amalgamated with the target bacterial cells to capture them. A magnetic field was employed to catch the nanobead-L. monocytogenes complex and the supernatants were removed. After a washing step, L. monocytogenes was separated from a food sample and was ready for detection. Results: The preliminary results showed that a capture and separation efficiency of 75% can be attained with 30 nm beads for L. monocytogenes in PBS solution, and the total immunoreaction time was 60 min. However, beads with larger diameter (150 nm) gave only ~ 40% capture efficiency, indicating that the developed immunomagnetic nanobeads (with diameter 30 nm) based separation method is beneficial over methods using beads with larger size because of their higher capture efficiency, minimal sample preparation, no need for mechanical mixing, and less background noise in detection. Further work will be concentrated on optimization of conditions of the immunomagnetic separation method and detection of the separated L. monocytogenes cells using microfluidic chip based biosensor. Significance: The outcome of this study will tremendously enhance the separation efficiency of foodborne pathogens, which will provide better prepared samples for further testing. P2-98 Susceptibility to Enterobacter sakazakii Changes with Increasing Age in Neonatal Mice ARENA N. RICHARDSON, Elizabeth A. Pollak, Denita Williams, Kwaku Agyekum and Mary Alice Smith University of Georgia, Environmental Health Science, Athens, GA, USA Introduction: Premature or very-low-birth-weight infants orally fed reconstituted powdered infant formula unintentionally contaminated with Enterobacter sakazakii (E. sakazakii) may develop infections resulting in severe outcomes such as septicemia, necrotizing enterocolitis, meningitis, or death. Infants who recover from infection may have morbidities such as hydrocephalus, mental retardation, or developmental delays. Although increasing age appears to reduce susceptibility, it is not known at what age infants become less susceptible to E. sakazakii infection. Purpose: Our objectives were to compare the susceptibilities of neonatal mice of different ages to E. sakazakii infection and to identify biomarkers of infection. Methods: Timed-pregnant CD-1 mice were obtained and allowed to give birth naturally. Neonatal mice were orally gavaged at postnatal day (PND) 1.5, 5.5, and 9.5 with a single dose of vehicle or 104, 108, or 1011 colony-forming units (CFU) E. sakazakii strain MNW2 per ml reconstituted powdered infant formula. On post-treatment day 7, surviving pups were sacrificed and brains, livers, and ceca were excised and analyzed for the presence of E. sakazakii invasion. Results: E. sakazakii was isolated from brains, livers, and ceca of neonatal mice treated at PND 1.5 and 5.5 but not from those of pups treated at PND 9.5. E. sakazakii was more invasive in brains than in livers and ceca, with total isolations of 25.3%, 21.2%, and 19.7%, respectively. Mortality was observed in neonates treated at PND 1.5 at a total of 11.25%, whereas no deaths occurred in neonates treated at the older ages. Significance: Like human infants, neonatal mice show a time-dependent susceptibility to E. sakazakii infection, with resistance increasing with increasing age. Future work will further characterize the infection and provide a model to develop treatments and therapies for E. sakazakii infection in human infants. P2-99 Evaluation of the Spartan DX™ Real-time Portable PCR Analyzer Using TrimGen eQ-PCR™ STEC-stx1 and STEC-stx2 Detection Kits for Shiga-toxin Genes M. E. Perez-Munoz, T. STILES, S. Stephens, H. Doong, C. Harder, N. Arbour and J. E. Stratton University of Nebraska-Lincoln, Food Science & Technology, Lincoln, NE, USA Introduction: The Spartan DX™ is a 4-well, portable, inexpensive real-time PCR platform. An evaluation of TrimGen eQ-PCR™ Shiga toxin detection kits for stx-1 and stx-2 genes was performed on two Spartan DX™ real-time PCR analyzers, which combines the three conventional steps of PCR (denaturation, annealing, extension) into two. Purpose: Evaluation of the Spartan DX™ Analyzer and TrimGen Shigatoxin stx1 and stx2 detection kits was performed to determine their usefulness and reliability for laboratories requiring non-batched results. Methods: DNA isolation was performed on 30 select strains of shiga toxin-producing and non-producing bacteria using Qiagen® DNeasy Blood and Tissue kits. Stx-1 and stx-2 genes were then detected simultaneously on both Spartan DX™ instruments using primer and probe sets for each gene, along with positive and negative controls. Also, diluted DNA samples and inoculated ground beef samples (101-103 CFU/g) were analyzed to assess the instrument’s sensitivity. Results: Results showed that 23 strains were positive for both genes (22 E. coli O157:H7, 1 Y. enterocolitica), 3 were positive for stx-2 only (Shigella sonnei, Shigella flexneri, Citrobacter freundii), and 4 were negative for both genes (Hafnia alvei, E.coli O129(B):H11, E. coli O157:H7 stx1-/stx2-, generic E. coli). Results for the Yersinia and Shigella strains indicate possible cross-reactivity of kit reagents with these species. Comparison of the Ct values for each instrument revealed no significant differences (P > 0.05). The analyzers succeeded in detecting genes in DNA samples diluted by 6 logs, and in isolates recovered from ground beef samples inoculated with decreasing levels of STEC, indicating a high degree of sensitivity. Significance: Overall, the Spartan DX™ PCR analyzer was user-friendly, easy to learn, and results were obtained rapidly (one hour following DNA isolation and PCR prep). This instrument could potentially improve the diagnostic capabilities of smaller laboratories or help developing countries obtain much needed molecular detection technology. Poster 140 — Abstract Book

IAFP 2009 Program Book and Abstract Book

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