title: PCR-based ribosomal DNA detection technique for microalga (Heterosigma carterae) causing red tide and its application to a biosensor using labeled probe.
abstract: A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate-labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.
authors.affiliation: Research Centre for Advanced Science and Technology, University of Tokyo, Tokyo, Japan. ryoichi.asai@aist.go.jp
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Asai
authors.forename: Kozue
authors.initials: K
authors.lastname: Ootani
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nomura
authors.forename: Chikashi
authors.initials: C
authors.lastname: Nakamura
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebukuro
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Arikawa
authors.forename: Jun
authors.initials: J
authors.lastname: Miyake
authors.forename: Isao
authors.initials: I
authors.lastname: Karube
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D015342:DNA Probes / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D056890:Eukaryota / Q000235:genetics*; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020349:Surface Plasmon Resonance
chemical_list: D015342:DNA Probes; D004275:DNA, Ribosomal