Highly sensitive Escherichia coli O157:H7 detection in a large volume sample using a conical polymer tube chamber consisting of micro-glass beads

Abstract

In this study, we developed a method for the highly sensitive detection of viable pathogenic bacteria in a large volume sample by performing RNA concentration, amplification, and using fluorescently tagged capture probes in a single polymer chamber without transferring RNA samples from one chamber to another. Nucleic acids were extracted from Escherichia coli O157:H7 and loaded into glass micro-beads embedded in a conical polymer tube chamber. Nucleic acids were concentrated in the micro-tube in a low pH buffer (pH 5). The mRNA, which was adsorbed to the glass micro-beads, was amplified by Nucleic Acid Sequence Based Amplification in the same chamber at a relatively high pH (pH 8.9). The products amplified were measured using the hair-loop type detection probe with FAM and DABCYL, which was pre-mixed in the NASBA mater mixture. As a result, high sensitivity (100% for rain water and 60% for river water) with less than 10 viable E. coli O157:H7 in 100ml could be achieved within 4h using the simple method proposed in this study.