2.3. Materials and sample preparation
Ultrapure DNase/RNase-free DW (Bioneer®, Daejeon,
Korea) was used in all experiments. Crystal violet and other chemicals
for the reaction buffer were purchased from Sigma-Aldrich Corp. (St.
Louis, MO, USA) and used without further purification. Bstpolymerase and RNase inhibitor were purchased from New England Biolabs
Inc. (Beverly, MA, USA). Thermostable RNase H was purchased from
NanoHelix Co., Ltd. (Daejeon, Korea). All oligonucleotides used in the
study (Table S1) were synthesized and purified by Integrated DNA
Technologies, Inc. (Coralville, IA, USA). Outer primers were purified by
polyacrylamide gel electrophoresis (PAGE), while inner primers and
cycling probe technology (CPT) probes were purified by high-performance
liquid chromatography (HPLC). The target bacteria, Escherichia
Coli O157:H7 were provided by Dxgene, Inc. (Seoul, Korea). The bacteria
were first cultivated at 37ºC for 16 h, and the colony-forming unit
(CFU) was measured using plate count agar (PCA) (NaraeBiotech, Inc.,
Gunpo, Korea). Based on the determined CFU, the culture solution was
diluted to 2.0 × 108 CFU/mL, which was then
centrifuged at 11,000 rpm for 5 min (Centrifuge 5424, Eppendorf,
Hamburg, Germany). The resulting precipitant was resuspended in the same
volume of Tris-HCl buffer (10 mM, pH 8.8), which was then subjected to
the serial dilution on the centrifugal microfluidic disc.