Specificity of a fluorogenic DNA probe for species of a Vibrio core group

Abstract

Specificity of a DNA probe developed for some species of a Vibrio core group was examined using a fluorescence resonance energy transfer system. The probe was designed based on the sequence of a 16S rDNA fragment of Vibrio parahaemolyticus cloned in a plasmid, pTPR1. The 5'-and 3'-end of the probe was ligated with fluorogenic dyes. The specificity was examined against clonal 16S rDNA fragments and chromosomal DNAs extracted from different species of Vibrio. The 16S rDNA fragments, cloned from V. parahaemolyticus, Vibrio natriegens, Vibrio fischeri, Vibrio mediterranei, and Vibrio tubiashii, had from one to four nucleotide substitutions compared with pTPR1 in the probe region. Probe hybridization with the clonal or chromosomal DNA fragments was monitored by determining the increase in fluorescence during PCR amplification. At the highest stringent condition, the probe hybridized only to identical sequences, but not to the sequences with substitution.