PCR-Based Ribosomal DNA Detection Technique for Microalga (Heterosigma carterae) Causing Red Tide and Its Application to a Biosensor Using Labeled Probe
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.
Keywordssmall subunit rDNA Heterosigma carterae fluorescence polarization asymmetric polymerase chain reaction surface plasmon resonance
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