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.