Purpose: A rapid test for the detection of foodborne pathogens like Salmonella, Campylobacter, Listeria and Cronobacter spp., and counting of Legionella in water, including the most relevant species, L. pneumophila was needed. The test is needed for early detection of pathogens, and for better and economical quality control.
Methods: The method is based on the detection of rRNA via hybridization events and specific capture and detection probes. Specificity is achieved by targeting conserved or unique rRNA sequences. A biotin-labeled capture probe is used to immobilize the target sequence on a solid support plate (streptavidin-coated microtiter plate). A digoxigenin-labeled detection probe provides an enzyme-linked optical signal read out. Detection results from application of anti-DIG-horseradish peroxidase Fab fragments. The bound complex is visualized by horseradish peroxidase substrate TMB (3,3’,5,5’-tetramethylbenzidine). Photometric data are measured at 450 nm and compared with standard solutions. The HybriScan software enables easy measurement and data analysis.
Results: Food samples were analyzed with the method and compared to the culture based method according to 64-LFGB. Five different food categories were tested. 355 food samples were analyzed and compared to culture-based method according to 64-LFGB. Validation was according to ISO 16140:2003 (ASU L00.00-22). Results of validation showed a relative accuracy of 99.2 %, relative specificity of 98.5% and relative sensitivity of 99.6%.
Significance: The new method is an economical, high throughput, 96-well microplate format system. The test is performed in less than 3 hours (in addition to the prep time) and offers significant time saving compared to cultivation-based assays.