Food Protection Trends - February 2010 - 104

Results: For 110 manipulated single cells growth was detectable in 79 samples (71.5%) with a final optical density of 1.21 × 109 CFU/ml (± 9.07%). In 31 samples (28.5%) growth was not detectable. the live/dead ratio of the initial culture was 20.9% (± 20.6%) as obtained by live/dead staining. these results show a good correlation of live/dead ratios before and after the sBCM indicating the ability of autonomous growth. Conclusions: these data suggest that the investigated single bacterial cells are able to multiply independently under optimal conditions. P2-03 Micrococcus roseus and Serratia marcescens as Coloured Bacterial Indicators: A Simple Strategy during Design and Development of a New Method for Sample Pre-treatment PeteR RossManItH, Karin Frühwirth, Beate süß, erich schopf and Martin Wagner, Dept. of Veterinary Public Health and Food science, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, austria Introduction: In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during setup and optimisation of an isolation process of bacteria, designed for food samples. Rational: Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. therefore, red-coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique. Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalisation of the method protocol. Prior to application, the chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g., centrifugal forces) and chemical (e.g., lysis buffer composition) conditions required during establishment of the new technique. Results: the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods. Conclusions: the use of these pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. the presumptive bacterial targets can be allocated simply by visualisation of their bright red colour silhouetted against the background sample matrix. application of coloured bacterial indicators saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession. P2-04 Statistical Data Analysis of Real-time PCR Results Derived from Single Copy Amplification PeteR RossManItH and Martin Wagner, Christian Doppler Laboratory for Molecular Food analytics, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, austria Introduction: the validation of real-time PCR systems and above all the proof of the detection limit of this method is a frequently and intensively discussed topic. We present a statistical method for the accurate determination of Dna amounts < 10 target molecules using real-time PCR. the implication of this method is the possibility of distinct validation of real-time PCR assays and the generation of absolute Dna standards needed for quantification with this enzymatic method in routine diagnostics. Rational: the purpose of this study was to evaluate a novel validation tool for real-time PCR assays based on the theoretical possibility of the amplification of one single Dna target. the ability to detect such low Dna target concentrations reliable by real-time PCR should be clearly demonstrated. Consequent a validation method based on this pre-requisites should be established which allows the absolute evaluation of real-time PCR assays. Real-time PCR was carried out by targeting a 274 bp fragment of the prfa gene of L. monocytogenes. Fit of the empirical data to the theoretical predictions was tested using the Kolomogorow – smirnov (K-s) test using the sPss 14.0 statistical software package. Results: the ability of the prfa real-time PCR assay to detect reliable one target molecule could be clearly demonstrated (pavg.=0.52). the coherence of the results of samples containing < 10 target molecules and samples containing Dna amounts within the range of fluorescent measurement could be clearly demonstrated. the evidence for the accuracy of the newly developed validation-method was shown both statistically and with direct demonstration. the explicit determination of assays with a detection limit of one copy and assays with such a limit of three copies is exemplary demonstrated. We also demonstrate that real-time PCR at best starts from the first cycle with certain efficiency and proceeds with this efficiency until saturation of the reaction. Conclusions: the results show that an absolute validation of real-time PCR assays is possible. the Ct values of certain initial target amounts are fixed in dependence of the efficiency of the reaction. an absolute determination of Dna amounts is possible independent of conventional measurement methods. the validation tool also allows on-line monitoring of real-time PCR results in routine diagnosis. 104 FOOD PROTECTION TRENDS | FEBRUARY 2010

Food Protection Trends - February 2010

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