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

• Published in: Microbes and Environments Vol. 18; no. 4; pp. 188 - 195
• Main Authors: Maeda, Toshimichi, Sasaki, Makoto, You, Mu, Ohsugi, Shin-ya, Mitsutani, Atsushi, Furushita, Manabu, Shiba, Tsuneo
• Format: Journal Article
• Language: English
• Published: Miyagi Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles 2003; Japan Science and Technology Agency
• Subjects: 16S rDNA; 5'-Nuclease PCR assay; Algal-lytic bacteria; Algicidal bacteria; Cytophaga; Marine; Skeletonema costatum; INW, Japan, Seto Naikai Sea, Hibiki-nada; INW, Japan, Honshu, Yamaguchi Prefect., Yoshimi Bay; INW, Japan
• ISSN: 1342-6311; 1347-4405
• DOI: 10.1264/jsme2.18.188

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 1010 molecules. The assay was applied to DNA extracted from natural seawater of Yoshimi Bay, Hibiki-nada 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.