Probe qpcr mix

For 10 s, #15 paper points were put in the gingival sulcus of four sites of two maxillary teeth (anterior and posterior tooth) and two mandibular teeth (anterior and posterior tooth) of subjects. The collected paper points were placed in sterilized tubes and stored instantly at −20 °C until just before analysis. DNA was extracted from collected #15 paper points using the AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, Korea) following the manufacturer’s instructions. OligoMix (YD Global Life Science Co., Ltd., Seongnam, Korea) and three types of oligonucleotides (Table 1) were used, which react particularly to each bacterium [19 ]. We combined 9 µL of OligoMix, 10 µL of 2× probe qPCR mix (Takara Bio Inc., Shiga, Japan), and 1 µL of template DNA to prepare a sample of polymerase chain reaction (PCR) reaction. The conditions of the PCR were as follows: initial denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, and annealing for 30 s at 62 °C. The above cycles were repeated 40 times. The Bio-Rad CFX Manager Software (Bio-Rad CFX Manager 3.1, Bio-Rad Laboratories, Hercules, CA, USA) program was used to calculate the cycle threshold (Ct) parameter, and the number of copies was derived by plotting the Ct value in the standard curve of each bacterium.

Quantification of eDNA was also performed by microfluidic dPCR using the BioMark Real-time System and 12.765 Digital Array (Fluidigm Corporation, South San Francisco, CA, United States) as previously described^{13 (link)}. For each sample, the PCR mixture (6 µL) contained 2 × Probe qPCR mix (3.0 µL; Takara Bio Inc.), 20 × binding dye sample loading reagent (0.6 µL; Fluidigm Corporation), forward and reverse primers (900 nM), TaqMan probe (125 nM), ROX solution (0.015 µL), and sample DNA (1.0 µL). We used three sets of primers and probes to quantify the eDNA of H. neglectus, O. latipes, and M. anguillicaudatus (Table 1). PCR was initiated at 98 °C for 2 min, followed by 50 cycles of 98 °C for 10 s and 60 °C for 1 min. The amplification curves obtained from individual reaction chambers of the microfluidic chip were analysed using Fluidigm Digital PCR analysis software (Fluidigm Corporation) to obtain abundance of DNA molecules.

Real-time PCR was carried out using a StepOnePlus Real-time PCR system (Applied Biosystems) and Probe qPCR Mix (Takara Bio). The reaction mixture was made in a final volume of 20 µL containing 10 µL of Probe qPCR Mix, 0.3 M of each primer^{10 (link)}, 0.1 M TaqMan probe^{10 (link)}, 0.2 M ROX reference dye and 2 µL of template DNA. PCR was carried out under the recommended conditions (95 °C for 30 s, (95 °C for 5 s and 61 °C for 30 s) × 45 cycles). As a negative control, distilled water was used instead of template DNA. The genomic DNA of P. infestans was adjusted to 0.01, 0.1, 1, 10, 100 and 1000 pg/µL TE using a spectrophotometer (BioSpec-nano, Shimadzu) and used as standards. The threshold was determined automatically by StepOne software v.2.1.