2.4. Detection of Spiroplasma in Ticks

To detect Spiroplasma DNA, PCR amplification of a sequence of approximately 1028 bp in the 16S rDNA was performed. The PCR was carried out in a 20 μL reaction mixture containing 10 μL of 2× Gflex PCR Buffer (Mg²⁺, dNTP plus), 400 nM of Tks Gflex™ DNA Polymerase (Takara Bio, Shiga, Japan), 400 nM of each primer, 1 μL of DNA template, and sterilized water. The reaction was performed at 94 °C for 1 min, followed by 45 cycles at 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 45 s and a final step at 68 °C for 5 min. PCR products were electrophoresed on a 1.0% agarose gel. The DNA of a Spiroplasma species isolated from I. persulcatus in our previous study [23] and sterilized water were included in each PCR run as positive and negative controls, respectively. Primer sets used for each assay are shown in Table 1 [13,36]. The amplified PCR products were purified using ExoSAP-IT Express PCR Cleanup Reagent (Thermo Fisher Scientific, Tokyo, Japan). Sanger sequencing was performed using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the ABI Prism 3130xl Genetic Analyzer according to the manufacturer’ s instructions. Sequence data were assembled using ATGC software version 6.0.4 (GENETYX, Tokyo, Japan).

Primers used in the present study.

NA, not applicable.