Highly sensitive Escherichia coli O157:H7 detection in a large volume sample using a conical polymer tube chamber consisting of micro-glass beads.
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.
Won
Ji Yeong
JY
Department of Chemical and Biomolecular Engineering, Sogang University, Mapo-gu, Seoul, Republic of Korea.
Min
Junhong
J
D058491:Bacterial Load / Q000295:instrumentation*; D015374:Biosensing Techniques / Q000295:instrumentation*; D018929:Cell Culture Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005898:Glass; D008863:Microspheres; D018401:Sample Size; D013048:Specimen Handling / Q000295:instrumentation; D014871:Water Microbiology; D014873:Water Pollutants / Q000302:isolation & purification*
D014873:Water Pollutants