Development of a LDR-microarray based diagnostic kit for Salmonella spp

Salmonella is a major cause of food-borne illness in humans, and can be lethal for immunocompromised persons or newborn children. Chickens are thought to be important vectors of Salmonella. Almost one quarter of European broiler flocks has been found to be contaminated with Salmonella (1), with S. Enteriditis and S. Thyphimurium being the most frequently isolated serotypes from broilers and clinical cases. Although immunoenzymatic detection techniques, which rely on the recognition of membrane antigens, have been the reference methods for many years, nucleic acid-based techniques, such as the Polymerase Chain Reaction (PCR) are currently replacing them. PCR and Nucleic acid-based techniques in general seem to be less expensive and less time consuming, and can directly investigate DNA regions affecting pathogenicity, although their specificity and sensitivity can still be improved. Here we present a new Salmonella diagnostic method based on the highly specific Ligase Detection Reaction (LDR), followed by hybridization of the reaction product on a very sensitive Universal Array (UA), that assures high sensitivity (Box 1). The LDR is preformed on the PCR amplification product of two Salmonella genes strongly associated with pathogenicity (fimA and invA). In order to build reliability, we sequenced this two genes from about 100 strains belonging to 60 serotypes. Moreover, for most of the Salmonella strains, and for all the most frequently isolated serotypes, more than one probe was designed. The diagnostic method was validated (Box 2) with (I) a specificity assay testing the discriminatory capability of the thermostable ligase, and (II) with a sensitivity test detecting as little as 1 fmol of PCR product. Furthermore, we succeeded in detecting Salmonella from artificially contaminated food at a concentration of 2 CFU/30g poultry meat.