Mupid exu

The genomic DNA (gDNA) or cDNA was amplified in a 20-μL PCR sample containing 1 U of i-StarTaq DNA polymerase (Intron Bio, Seongnam, Korea), 1× PCR buffer (30 mM Tris-HCl (pH 9.0), 30 mM salts containing of K⁺ and NH₄⁺, 2 mM MgCl₂), 2 mM dNTP mix, and 10 pM of each gene-specific primer. PCR conditions were pre-denaturation at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s, followed by 72 °C for 5 min. PCR products were analyzed by gel electrophoresis (Mupid-exU; Takara Bio) at 100 V for 20 min on a 1.25% agarose gel with RedSafe (iNtRON Biotechnology). Gel images were obtained using a Bio-1000F gel imager (Microtek International, Hsinchu, Taiwan, Republic of China). All of the primer sequences are presented in Table 2. RN18s is used as control. RN18s is a housekeeping gene like GAPDH and has been reported to be very stable gene in pigs [28 (link)].

PCR analysis was conducted to determine the presence of specific Burkholderia clades in total DNA extracts from insect and soil samples. First, all samples were subjected to PCR analysis to detect the presence of genus Burkholderia using Burkholderia-specific 16s rRNA primers (Table 1).
Then, Burkholderia positive samples were subjected to subsequent PCR analysis to determine the Burkholderia clade composition in the samples using clade-specific primer sets including BCC, PBE, and SBE clades (Table 1) with temperature profile of 94°C for 5 min followed by 35 cycles of 94°C for 30s, 55°C for 1 min, and 72°C for 2 min [26 (link),35 ]. Gel electrophoresis was performed to confirm the presence of target primers on a 1.0% agarose gel slab containing Tris-acetate-EDTA buffer (40 mM Tris-Acetate, 1 mM EDTA, pH 8.3) at 120 v/cm (Mupid-exU, Takara Bio Inc., Shiga, Japan) followed by visualization on a digital gel documentation system. Burkholderia positive samples that did not belong to any of the three clades were defined as unclassified in this study.

All specimens containing M. pneumoniae DNA had been stored at −70 °C before testing. Nucleic acid extraction was performed using the Ribospin TM vRD plus kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. The extracted DNA was subjected to PCR using the Pfu Plus 5× Master mix (Elpis Biotech, Daejeon, Korea) according to the manufacturer’s instructions. The primers targeted residues 1998–2018 (forward) and 2673–2692 (reverse) in domain V of 23S rRNA (Myco23S-F: 5-TCTCGGCTATAGACTCGGTGA-3 and Myco23S-R: 5-TAAGAGGTGTCCTCGCTTCG-3).
The PCR products were subjected to 1% agar gel electrophoresis using a submarine-type electrophoresis device (Mupid-exU; Takara Bio Inc., Shiga, Japan). The approximately 700-bp band was identified under ultraviolet light, photographed using an imaging system (Geldoc XR image system; BioRad, Hercules, CA, USA), and then excised. The gel extraction procedure was performed using a commercial gel extraction SV kit (MGmed, Seoul, Korea), and the product was analyzed using the BigDye^® Terminator v3.1 Cycle Sequencing and an ABI PRISM 3730XL Analyzer (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). The DNA sequences were evaluated for point mutations at residues 2063, 2064, and 2067 in domain V of 23S rRNA using a sequence that is registered in the GenBank database (GenBank number: X68422).