Each mosquito was homogenized in 300µl of saline buffer. The homogenate was centrifuged at 800g for 2min, and the supernatant was retained. Total DNA was extracted from 100µl of supernatant using the RiboPrep kit (Amplisense, Moscow, Russia) according to the manufacturer's instructions. PCR was carried out in a 25μl volume with an Encyclo Plus PCR kit (Evrogene, Moscow, Russia). The COI gene fragments were amplified using primers 1454F and 2160R and 2027F and 2886R according to the original protocol (25 (link)). The amplification products were sequenced with forward and reverse primers for each primers pair. Sequencing of amplicons was performed using an ABI PRISM 310 sequencer and a BigDye Termination kit as recommended by Applied Biosystems (United States). The sequences were edited manually with Chromas and then aligned using MAFFT v7 (https://mafft.cbrc.jp ). The total length of the COI gene sequences was 1317–1433bp. The nucleotide sequences were deposited in GenBank under accession numbers MZ 501500-MZ501561. Sequences MH817490–MH817558 from our preliminary study (38 ) were also included in the analysis. A total 131 sequences from individual mosquitoes were used for the genetic analysis.
Protocol detail
Mosquito DNA Extraction and COI Sequencing
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Buffer
Gene
Genetic
Mosquitoes
Nucleotide sequences
Primers
Prism
Saline
Corresponding Organization :
Other organizations : Vavilov Institute of General Genetics, Central Research Institute of Epidemiology
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Variable analysis
independent variables
- Homogenization method (300µl of saline buffer)
- Centrifugation (800g for 2min)
- DNA extraction method (RiboPrep kit)
- PCR amplification (Encyclo Plus PCR kit, primers 1454F and 2160R, 2027F and 2886R)
- Sequencing method (ABI PRISM 310 sequencer, BigDye Termination kit)
- Total DNA extracted
- COI gene fragments amplified
- COI gene sequence length (1317–1433bp)
- COI gene sequences deposited in GenBank (accession numbers MZ501500-MZ501561)
- Mosquito samples
- Manufacturer's instructions for DNA extraction and PCR amplification
- Not explicitly mentioned
- Not explicitly mentioned
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