As part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific forSalmonellaspecies were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43Salmonellaspp. and 47 non-Salmonellastrains. The most selective primer set was found to be 139-141 (K. Rahn, S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galán, C. Ginocchio, R. Curtiss III, and C. L. Gyles, Mol. Cell. Probes 6:271-279, 1992), which targets theinvAgene. An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for theinvAprimer set. To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with theinvAtarget gene, was constructed. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 10⁴CFU/ml was used as the template in the PCR (50 CFU per reaction). The primer set was further validated in an international collaborative study that included 16 participating laboratories. Analysis with 28 coded (“blind”) DNA samples revealed an analytical accuracy of 98%. Thus, a simple PCR assay that is specific forSalmonellaspp. and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard. This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data.

A simple and ready-to-go test based on a 5′ nuclease (TaqMan) PCR technique was developed for identification of presumptiveSalmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic “rough” isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonellastrains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [ΔRn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (ΔRn, ≤0.5). All 100 roughSalmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at −20°C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification ofSalmonella, particularly in large reference laboratories.

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

A number of intervention strategies against Campylobacter -contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter , and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold ( C _T ) values ( R ² = 0.993), with a quantification range of 1 × 10 ² to 1 × 10 ⁷ CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high ( R ² = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter . No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.