Takara la taq

An apterous adult aphid from each sample was washed three times in ultrapure water. Total genomic DNA was extracted from whole individuals with the DNeasy Blood & Tissue kit (QIAGEN) according to the manufacturer’s instructions. DNA extraction was carried out in an ultra-clean workbench to avoid contamination of environmental DNA. The bacterial universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [37 (link)] were used to verify the success and quality of the DNA extractions. To assure the accuracy of results, sterile deionized water was used as a negative control. PCR amplifications were performed in 25 μL reactions containing 1 μL of DNA, 2.5μL 10× LA PCR buffer II (Mg²⁺ plus), 0.5 μL dNTP mixture (2.5 mM each), 0.5 μL of each primer (10 μM), 0.5 μL of TaKaRa LA Taq (5 U/µL) (TaKaRa Bio Inc., Otsu, Japan), and 19.5 μL water. Amplification was performed in ProFlex^TM Base (Applied Biosystems, Inc., Waltham, United States), using the following cycling conditions: 94 °C for 4 min, followed by 30 cycles of 30 s at 94 °C, 40 s at 65 °C, 90 s at 72 °C, and a final extension of 10 min at 72 °C. The PCR products were detected on 1% agarose gels, and the positive samples with a bright band of about 1500 bp were kept at −20 °C until 16S library preparation. The negative controls had no bands.

Total RNA was extracted from 300 μl serum samples using QIAamp UltraSens Viral RNA kits (Qiagen, Hilden, Germany). RNA was reverse-transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). DNA was extracted from 400 μl PBMC samples using a QIAamp DNA Mini kit (Qiagen), and 5 μl aliquots of DNA were used for nested PCR [21 (link),22 (link)]. The env gene was amplified via nested PCR with TaKaRa LA-Taq (Takara Bio Inc., Shiga, Japan). The first and second PCR reactions were performed in reaction mixtures of 25 μl and 50 μl, respectively. The outer primer pairs were OWE1 and OWE2, whereas the inner primer pairs were OWE3 and OWE4 [21 (link),22 (link)]. The amplicons were directly sequenced using an Applied Biosystems 3730XL Analyzer (Foster City, CA, USA).

The analytical sensitivity of the Bicolor FMCA was estimated by serial dilution experiments using three recombinant plasmids p-B, p-N, and p-H. The three plasmids were generated using the pMD18-T Vector Cloning Kit (Takara, Dalian, China), and the insert fragments of the plasmids were amplified from Bartha-K61, NIA3, and HDDJ using primers wai3F/wai4R/wai4BR with the TaKaRa LA PCR Kit version 2.1, respectively. Each 25 μL PCR contained 1 × GC Buffer I, 400 μM of each deoxynucleoside triphosphate (dNTP), 0.4 μM primers wai3F/wai4R/wai4BR, 1.25 U of TaKaRa LA Taq (Takara, Dalian, China), and 2 μL of template DNA. Thermal conditions involved initial denaturation at 95 °C for 3 min, followed by amplification using 45 cycles of 95 °C for 40 s, 62 °C for 40 s, and 72 °C for 90 s. Each plasmid was 10-fold serially diluted with water to concentrations of 1 × 10⁹ copies/μL to 1 × 10⁰ copies/μL. The limit of detection for each genotype was determined from the lowest concentration measured by the Bicolor FMCA.

DNA extraction and PCR mixture preparation were performed on a clean bench to reduce contamination. DNA extraction from culture samples was performed as described previously^{53 (link)}. The concentration of extracted DNA was measured using a Quant-iT dsDNA High-Sensitivity Assay Kit (Life Technologies). PCR amplification was performed using the Takara Ex Taq (for conventional clone analysis) or Takara LA Taq (for Illumina-based amplicon sequencing (iTAG) for targeted sequencing for the SSU rRNA gene analysis) (Takara Bio), and the reaction mixtures for PCR were prepared according to the manufacturer’s instructions. For the conventional clone analysis, a universal primer pair 530F/907R^{51 (link)} and an archaeal primer pair 340F/932R^{15 (link),54 (link)} were used for PCR amplification. For iTAG analysis, the universal primer pair 530F/907R, which contained overhang adapters at the 5′ ends, was used. The procedures used for library construction, sequencing and data analysis were described previously^{21 (link),55 (link)}.

Based on the variations observed from the sequencing results, we confirmed that both opsin genes mws and lws were amplified together by the primer pairs used. In order to sequence the two genes separately, PCR products obtained from amplification of exon 5 from the two samples (A. seniculus and A. caraya), were cloned into plasmid vectors using TA cloning® kits (Invitrogen). Multiple clones of each sample were isolated, purified via spin columns (Qiagen), and sequenced using M13 vector primers. Based on sequences analyses from 10 individual clones of each sample, we were able to differentiate exon 5 of the mws and the lws genes, and to design specific primers for each gene. In an inter-exon PCR, we combined a common forward primer for both genes located in exon 3, with reverse primers specific for the mws and for the lws genes, located at exon 5 (Supplementary Table S1). The PCRs were performed using the enzyme TaKaRa LA Taq® (Takara Bio USA), and PCR parameters were based on Davidoff, Neitz & Neitz [39 (link)] (Supplementary Table S2). The PCR products over 2 kb were visualized using a 2% agarose gel, purified, and sequenced in both directions as described above. Sequences were analyzed with BioEdit 7.25 [38 ], and exons 3 and 5 of each gene were identified.

DNA extraction and PCR mixture preparation were performed under a KOACH T 500-F tabletop air filtration system with a static remover (KOKEN Ltd., Tokyo, Japan) to reduce contamination. To extract DNA from the samples, an ISOL for Beads Beating kit (Nippon Gene, Tokyo, Japan) was used according to the manufacturer’s protocol. The concentration of extracted DNA was measured using a Quant-iT dsDNA High-Sensitivity Assay Kit (Life Technologies, Carlsbad, CA, USA). PCR amplification was performed using TaKaRa LA Taq (TaKaRa Bio Inc., Kusatsu, Shiga, Japan), and the reaction mixtures for PCR were prepared according to the manufacturer’s instruction. For PCR amplification, a universal primer pair 530 F/907R^{81 (link)} was used to amplify SSU rRNA genes (V4-V5 regions). The PCR primers contained overhang adapters at the 5′ ends. PCR amplification conditions were as described previously^{82 (link)}. Detailed following procedures for sequencing and data analysis as described Supplementary Text 2.