Food Protection Trends - February 2010 - 103

the complete procedure can be executed within 7 hours, enabling a serotype result within one working day after isolation. the test is being used by a range of laboratories, including reference laboratories, and yields reproducible results after a 2-day training. the test has proven to be independent of the culture media used and is capable of typing rough Salmonella strains (in contrast to KW). the process to obtain external certificates is in progress: the last part for the oIe-validation is completed and the certificate is expected end 2009. the protocol for joint aoaC & MicroVal validation is being finalized. the Premitest Salmonella has been designed for food processors, as routine use. However, the set-up of the test enables the inclusion of specific Dna markers. In this way, more detailed knowledge on specific serotypes or characteristics (such as antibiotic resistance and the Dt104) can be obtained. Conclusions: the Premitest Salmonella is a good tool for official labs to get fast and reliable serotyping and the best routine tool for the food industry for serotyping Salmonella isolates. It is inevitable in surveillance and outbreak-related studies. Poster Session 2 – Friday, 9 October P2-01 Stability of Calibration Function (Standards) in Nucleic Acid-based Food-pathogen Detection BaRBaRa RoeDeR, Karin Frühwirth, Martin Wagner, and Peter Rossmanith, Christian Doppler Laboratory for Molecular Food analytics, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, austria Introduction: Results of real-time PCR depend on a calibration function when using the Ct-method for quantification and the accuracy of this standard is all-important. Long-term storage of standards is saving cost and time, avoiding laborious production on a daily basis in routine application. nevertheless, aqueous solutions of Dna are prone to degradation during storage. Rational: the aim of this study was the determination of the causative parameter of Dna-standard degradation and the underlying mechanism impairing the amplification reaction during real-time PCR. Real-time PCR assays targeting the prfa gene of Listeria and the fima gene of Salmonella have been used to investigate the influence of long-term storage (>100 d), the GC-value, shear forces, DNA target length, chemical reactions within the storage buffer, glycerolstorage, subsequent thaw and freeze cycles, and the influence of remaining cellular enzymes after isolation, at -20°C, ± 0°C, and 4°C. tests were performed for initial low and high Dna target numbers. Results: the stability of Dna-standards is influenced by shearing of long Dna fragments (bacterial genomes) if the standard is frozen. short fragments (~100 bp) are not influenced by shearing during long-term storage or by subsequent thaw and freeze cycles. Depurination of the Dna and following mismatches on the primer attachment sites are biasing real-time PCR results if Dna is stored at 4°C or with glycerol. this effect is increased by primary amines such as tris or by Mg-ions as included in the PCR buffer. By finally testing the resulting benchmark treatment for Dna-standard storage a deviation of 0.2 Ct-values was obtained by real-time PCR after 100 days storage in H2o containing 50% glycerol. Conclusions: Preservation of Dna-standards in 50% glycerol in ddH2o enables long-termstorage for real-time PCR. Depurination and shearing of the Dna are avoided, thus providing reliable results using the Ct-method for quantification. P2-02 Application of a Novel Single Bacterial Cell Manipulation Technique BaRBaRa RoeDeR, Martin Wagner, and Peter Rossmanith, Christian Doppler Laboratory for Molecular Food analytics, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, austria Introduction: Highly diluted suspensions of bacterial cells are distributed according to Poisson-distribution. Quantitative microbiological methods (Most propable number MPn) are based on this prerequisite. nevertheless, predictions on the growth performance of single bacterial cells are not possible due to these statistical effects. a new method avoiding these influences was developed which enables the physical manipulation of single bacterial cells. Based on this method the investigation of autonomous growth of low bacterial cell inocula (< 10) was performed. Rational: the purpose of this study was to evaluate the growth performance of single bacterial cells without the influence of statistics within dilution series. Furthermore the necessity of chemical and physical cell to cell interactions for bacterial growth was investigated. Listeria monocytogenes EGDe bacteria from the lag-phase, the mid-exponential phase, and the stationary phase were used to produce single cell inocula with the newly developed single bacterial cell manipulation technique (SBCM). Growth in tryptone soy broth with 0.6% yeast was evaluated after 24 hours at 37°C or 42°C by measurement of optical density and selective- and non selective plating. FEBRUARY 2010 | FOOD PROTECTION TRENDS

Food Protection Trends - February 2010

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