A colorimetric microtiter plate sandwich DNA hybridization system for the detection of Salmonella in foods

Abstract

A microtiter plate sandwich DNA hybridization system for the specific detection of Salmonella-contaminated food samples was developed. The capture probe of this system was a single strand plasmid M13 DNA with a 0.4 kb HindIII- EcoRI insert, while the detection probe was a 1.4 kb HindIII- EcoRI DNA fragment prepared from the replication form (RF) of a recombinant M13 DNA containing this 1.4 kb fragment. Labeling of the detection probe was performed by nick-translation using biotin-21-dUTP. Both the 0.4 kb and the 1.4 kb HindIII- EcoRI DNA fragments were derived from a 1.8 kb HindIII DNA fragment which under specific conditions could hybridize with all the Salmonella isolates tested but not with any of the non- Salmonella strains including Enterobacteriaceae, a species which is closely related to Salmonella. When the capture probe used for microtiter plate coating was 2 μg per well while the detection probe was 100 ng per well, target DNA obtained from 10 3 cells of Salmonella could be detected without ambiguity. When this system was used for the detection of Salmonella artificially added to food samples such as milk, pork and beef, the results showed that this system was reliable, although target DNA prepared from one ml of the mixture containing N × 10 6 cells was required for each microtiter well to yield a positive reaction. Examination of the storage stability of the microtiter plates precoated with capture probes showed that within 16 d of storage at 8°C, these microtiter plates were reliable for the detection of Salmonella.