Premix taq

A total of ten species in the Hyrcanus group distributed in China were collected; these were An. belenrae, An. crawfordi, An. junlianensis, An. kleini, An. kunmingensis, An. lesteri, An. liangshanensis, An. peditaeniatus, An. sinensis, and An. yatsushiroensis. The specimens were collected from ten sites in seven provinces in China between 1997 and 2015. Adult mosquitoes were collected by mosquito light traps (Houji Dianzi, Shen Zhen, China) or mosquito aspirators (Table 1). The collected adult mosquitoes were killed with chloroform vapor, dried, and stored separately in 1.5 mL centrifuge tubes with a desiccant. Mosquitoes were brought back to the laboratory and identified based on morphological characteristics as Hyrcanus group species and stored at −80 °C until genome extraction. After extraction of genomic DNA, the species identifications of all samples were confirmed by amplifying the ITS2 sequence using PCR [26 (link)]. The reaction mixture is 20 μL containing genomic DNA as templates 10 μL premix Taq (Aidlab, Beijing, China), 0.5 μL forward and reverse primers each, 1 μL genomic DNA and 8 μL ddH₂O. The PCR reaction was conducted in a ProFlex PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA), and the cycling conditions were 94 °C for 2 min, followed by 30 cycles of 94 °C/30 s, 45 °C/30 s, 72 °C/30 s, with a final extension at 72 °C for 5 min.

The VGSC gene of Ae. albopictus contains four domains (I–IV), and nonsynonymous point mutations have been found only in Domain II, III, and IV [17 (link), 32 (link)]. To detect point mutations in VGSC, fragments from Domain II, III, and IV were amplified via PCR under the same conditions. The 20 μL reaction mixture contained genomic DNA as templates, 10 μL of premix Taq (Aidlab, Beijing, China), 0.5 μL each of forward and reverse primers, 1 μL of genomic DNA, and 8 μL of ddH₂O. The PCR reaction was conducted in a ProFlex PCR System (Applied Biosystems, Thermo Fisher Scientific), with the following cycling conditions: 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 68°C for 30 s, with a final extension at 68°C for 8 min. The amplified products were detected using 1.5% agarose gel electrophoresis and sequenced using the Sanger sequencing method by BioSune Biotechnology Co., Ltd. The amplification and sequencing primers were the same and are listed in Table 2 [17 (link)].
To distinguish the aberrant peaks in the sequence data, TA cloning was performed on the PCR products of several individuals for Domain III using a pMD 18-T Vector Kit (TaKaRa, Dalian, China) following the manufacturer’s instructions for cloning and sequencing.