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 ZolTM 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 DiastarTM 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.
To detect the partial SFTSV M-segmented gene, a one-step reverse transcription PCR (RT-PCR) was performed using a DiastarTM 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.
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