Accupower hotstart pcr premix kit

Genomic DNA was extracted from whole blood samples using the DNeasy Blood and Tissue Kit (Qiagen, Melbourne, Australia) according to the manufacturer’s protocol, and its quantity and quality were measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) before storage at − 20 °C until analysis.
Screening was performed by nested PCR using the AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Korea) and designated primer sets. Anaplasma spp. were detected based on amplification of the 16S rRNA gene using the primer sets EE1/EE2 and EE3/EE4 [19 (link)]. Borrelia spp. were identified based on the presence of the 5S (rrf)–23S (rrl) intergenic spacer using primer sets Bb23S3/Bb23Sa and Bb23SnF/Bb23SanR, and B. burgdorferi was detected by amplification of the outer surface protein A gene fragment using primer sets N1/C1c and N2/C2c [20 (link)]. Hemoplasmas were first identified based on the amplification of 16S rRNA with universal primers fHf1/rHf2 and M. suis-specific primers f2/r2 [16 (link), 21 (link)]; positive results were then confirmed at the species level by PCR using cmsf2/cmsr2 and msf2/msf2 primer sets to amplify the 16S rRNA gene of M. suis, M. parvum, and the novel hemotropic M. haemosuis [12 (link)].

Approximately half of the collected ticks were used to detect SFTSV infection, and the remainder were stored in 70% ethanol for long-term use as a biological resource. The tick pools (1–5 for adults, 1–30 for nymphs, and 1–50 for larvae) were homogenized by species, survey period, and collection site using a Precellys^® CK28-R Lysing kit (bead tube for hard tissue homogenization; Bertin Technologies, Bretonneus, France) and Precellys^® evolution (homogenizer; Bertin Technologies, Montigny-le-Bretonneux, France) followed by total RNA extraction from the pools using a commercial Direct Zol^TM RNA extraction kit (Zym Research, Irvine, CA, USA) with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturers’ instructions.
To detect the partial SFTSV M-segmented gene, a one-step reverse transcription PCR (RT-PCR) was performed using a Diastar^TM 2× OneStep RT-PCR premix (SolGent Co., Daejeon, Korea) with SFTSV-specific MF3 (5’-GATGAGATGGTCCATGCTGATTCT-3’)/MR2 (5’-CTCATGGGGTGGAATGTCCTCAC-3’) primers [3 (link)]. Afterwards, nested PCR was performed using an AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Korea) with SFTSV-specific MMF3 (5’-TAAACTTGTGTCGTGCAGGC-3’)/MMF2 (5’-CCCAGCGACATCTCCTTACA-3’) primers [3 (link)]. The minimum infection rates (MIRs, number of positive pool of mites/total number of ticks tested × 100) were then calculated.

Nucleic acid was manually extracted from a blood sample using the DNeasy Blood and Tissue Kit (QIAGEN, Germany) according to the instruction and it`s stored at -20°C. We used nested AS-PCR to detect MMP9 rs3918242 genotypes [28 (link)]. Also, RFLP was used to detect the variations of rs1799750 of MMP1, rs11646643 and rs243864 of MMP2, rs3918253 of MMP9, rs652438 of MMP12 gene [29 (link)–31 (link)]. List of the primers, restriction enzymes and length of amplicons are shown in S1 Table. PCR reactions were performed using the AccuPower® HotStart PCR PreMix Kit (K-5050, Bioneer Corporation, Korea). The amplicons and their length were determined by agarose gel electrophoresis (C-9100-1, Bioneer Corporation, Korea) and visualized with ethidium-bromide staining (C-9036, Bioneer Corporation, Korea).

Total RNA was extracted with TRIzol reagent according to the manufacturer’s instructions. Total RNA (1 µg) in the final 20 µL cDNA system was synthesized by reverse transcription synthesis kit (#04897030001; Roche Diagnostics, Mannheim, Germany). A semi-quantitative reverse transcriptase–polymerase chain reaction (sqRT–PCR) was performed using an AccuPower HotStart PCR PreMix kit according to the manufacturer’s instructions (K-5051; Bioneer, Daejeon, Korea) in a MyGenie 96 Thermal Block (Bioneer, Daejeon, Korea). The cycle sequence of each PCR reaction was performed using an initial denaturation at 94 °C for 5 min, followed by 28 cycles at 94 °C for 30 sec, 52 °C for 30 sec, and 72 °C for 30 sec. After 28 cycles, an additional elongation step was performed at 72 °C for 7 min. PCR products were identified by electrophoresis with 1.0% agarose gels and recorded using a Gel Doc 1000 imaging system (Bio-Rad, Carlsbad, CA, USA). GAPDH RNA served as a housekeeping gene relative to the control.