Eurotox 2003

As part of a major international project for the validation and standardization of PCR for detection of five major foodborne pathogens, four Salmonella -specific primer sets were evaluated in-house with respect to their analytical accuracy (selectivity and detection limit) on 43 Salmonella and 47 non- Salmonella strains. The most selective primer set was found to be 139/141 published by Rahn et al., 1992, which targets the invA gene. An extended determination of the selectivity using 364 strains showed that the inclusivity was 99.6% and the exclusivity 100% for the invA primer set. For the indication of possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC) was constructed, which is co-amplified with the invA target gene. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139/141 was found to be 100%, when using a 10¶4 CFU/ml cell suspension as template in the PCR reaction (50 CFU per reaction). The primer set was further validated in an international collaborative study participating 16 laboratories. Analysing 28 coded "blind" DNA samples resulted in an analytical accuracy of 98%. The performance of a PCR method for the detection of Salmonella on artificially inoculated chicken-rinse and pork swab samples was conducted in a second ring-trial participating 15 laboratories. The diagnostic specificity, sensitivity, accordance (repeatability) and concordance (reproducibility) was 100% for the pork samples. For the chicken-rinse samples, the sensitivity, accordance and concordance was also 100% and the specificity of 87.2%. Thus, a simple PCR method specific for Salmonella spp., which amplifies a chromosomal DNA fragment detected by gel electrophoresis, is established through extensive validation and is currently proposed as international standard document. The work done in this study can contribute to meet the increasing demand of quality-assured laboratories for standard diagnostic methods and to facilitate international comparison and exchange of epidemiological data.