The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature ( T _m ) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T _m , which was consistently specific for the amplicon obtained; the mean peak T _m obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10 ³ to 10 ⁸ CFU/ml) showed good linearity ( R ² = 0.9767) and a sensitivity limit of less than 10 ³ CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.

An international outbreak linked to oyster consumption involving a group of over 200 people in Italy and 127 total subjects in 13 smaller clusters in France was analyzed using epidemiological and clinical data and shellfish samples. Environmental information from the oyster-producing area, located in a lagoon in southern France, was collected to investigate the possible events leading to the contamination. Virologic analyses were conducted by reverse transcription-PCR (RT-PCR) using the same primer sets for both clinical and environmental samples. After sequencing, the data were analyzed through the database operated by the scientific network FoodBorne Viruses in Europe. The existence of an international collaboration between laboratories was critical to rapidly connect the data and to fully interpret the results, since it was not obvious that one food could be the link because of the diversity of the several norovirus strains involved in the different cases. It was also demonstrated that heavy rain was responsible for the accidental contamination of seafood, leading to a concentration of up to hundreds of genomic copies per oyster as detected by real-time RT-PCR.

The explosive increase since the beginning of 1990s in the number of publications reporting PCR-based methods for de- tection or molecular typing of food-borne pathogens has at- tracted the attention of end user laboratories. However, the well-recognized difficulties in reproducing published tests due to variation in the performance of PCR thermal cyclers (7) and in efficiencies of different DNA poly- merases and to the presence of PCR inhibitors in the sample matrix have hampered implementation in end user laborato- ries. This particularly applies to laboratories with quality as- surance programs. It is necessary to have PCR-based guidelines available as internationally recognized standards (5). Currently, the lack of international standards often forces end user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that end users and reference laboratories have ac- cess to open-formula, noncommercial, and nonproprietary PCRs in which the information on target gene and reagents is fully available. The prerequisites for a PCR published in the scientific lit- erature to be adopted as a standard are that it has to be nonproprietary and that it has to have been validated through a multicenter collaborative trial according to the international criteria (1, 2, 5). Multicenter trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOOD- PCR (http://www.pcr.dk)) involving 35 laboratories from 21 countries (4, 6). A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube which is coamplified simul- taneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But it could also mean that the reaction was inhibited due to malfunction of the thermal cycler, incorrect PCR mixture, poor polymerase activity, and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target se- quence present. When neither IAC signal nor target signal is produced, the PCR has failed. Thus, when a PCR-based method is used in routine analysis, an IAC, if the concentration is adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by retesting the sample. The European Standardization Committee, in collaboration with International Standard Organization, has proposed a gen- eral guideline for PCR testing that requires the presence of IAC in the reaction mixture (3). Therefore, only IAC-contain- ing PCRs may undergo multicenter collaborative trial, which is a prerequisite for standardization. The scientific journals must provide the source of new PCR- based methods suitable for standardization. Therefore, we pro- pose that henceforward the editorial boards of applied micro- biology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be done by

Clostridium botulinum types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the C. botulinum subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including C. botulinum types C (n = 12), C-D (n = 29), D (n = 5), and D-C (n = 10), other botulinum neurotoxin (BoNT)-producing Clostridium strains (n = 20), non-BoNT-producing clostridia (n = 20), and other bacterial species (n = 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of C. botulinum in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n = 108), poultry (n = 60), and bovines (n = 56). Among the 292 samples, 144 were positive for either the bont/C-D or the bont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.