Japanese Journal of Food Microbiology
Online ISSN : 1882-5982
Print ISSN : 1340-8267
ISSN-L : 1340-8267
Development of a Rapid Salmonella Detection Kit by the Colorimetric DNA/RNA Hybridization Method in Microtiter Wells
Takanori NAMIMATSUMika TSUNAYasuo IMAISatoshi FUTOShizuo MITSUSETetsuya SAKANOShizuo SATO
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1998 Volume 15 Issue 1 Pages 47-53

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Abstract

We developed a rapidSalmonelladetection kit using the colorimetric DNA/rRNA solid phase sandwich hybridization method in microtiter wells. This sandwich hybridization requires two adjacent, non-overlapping probes, one fixed to a well capture probe (Capture) and another to a labeled detection probe (Detector). The DNA sequence in the 23S rRNA gene of Salmonella Typhimurium was determined. By comparison of this sequence with those ofEscherichia coli, Citrobacter freundii and Enterobacter cloacae, two oligonucleotide probes (Capture and Detector) for the detection kit were selected. The OD value of the substrate reaction was highest when the non-target region between Capture and Detector wasO base. When HTT broth was used for the selective enrichment broth, sandwich hybridization reaction was inhibited by the deposited calcium on the microtiter well bottom . EDTA (final concentration 133mM) was the most effective chelate agent which inhibited deposition of calcium to the micro-titer plate bottom but not the hybridization reaction forSalmonellaTyphimurium. The kit effectively identified all 12 serovars ofSalmonellaspp . tested, and yielded no false-positive reactions in the examination of 15 pure cultures of non-salmonella.

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