16S rRNA Sequence-based Rapid and Sensitive Detection of Aquatic Bacteria by On-chip Hybridization Following Multiplex PCR

Abstract

We applied transcription in vitro following multiplex PCR in order to improve the sensitivity and rapidity of microbial detection in aquatic environment with an oligonucleotide microarray. Transcripts of 16S rRNA gene were fluorescently labeled and hybridized on fabricated oligonucleotide chip. The assay sensitivity was evaluated by detecting cultured bacteria inoculated into natural river water. By using our procedure, the assay was completed more rapidly (6 hr) than conventional oligonucleotide microarray assays (>12 hr), and its sensitivity was improved: detection limit was decreased by one order of magnitude. This method might be useful for monitoring pathogenic bacteria in aquatic environments.