5'-Nuclease PCR Assay of Gliding Bacteria that Kill Skeletonema costatum in Seawater.

Abstract

A 5'-nuclease PCR assay, targeting 16S rDNA, was developed to detect a group of gliding bacteria that digest Skeletonema costatum cells and are phylogenetically close to Cytophaga latercula. The detection limit was 15 molecules of the target DNA in one reaction mixture. The assay is so strict that the probe did not hybridize to DNA fragments with one nucleotide mismatch, even though the amount of DNA fragments was increased to the order of 10 10 molecules. The assay was applied to DNA extracted from natural seawater of Yoshimi Bay, Hibikinada Sea, Japan, during the period from August 1998 to February 1999. A positive result was obtained only for a seawater sample of September 10, 1998. Among 30 PCR clones obtained from the 5'-nuclease PCR product of the positive sample, 25 clones gave positive results and 4 clones negative results in the assay. The positive clones examined were identical in the structure of the probe region, whereas negative clones had one nucleotide mismatch or deletion. The results indicate that the assay detects only the target sequence even in natural seawater DNA.