关键词: 中华按蚊； kdr基因； 基因突变； TaqMan?MGB探针； 实时荧光定量PCR

Abstract:

Objective To establish a Real?time Fluorescence Quantitative PCR to detect the kdr gene mutation in Anopheles sinensis. Methods One pair of primers and three TaqMan?MGB probes were designed based on kdr gene and its L1014 locus mu? tations of A. sinensis. After optimization，the Real?time Fluorescence Quantitative PCR was verified by using 6 types of A. sinensis samples with different kdr gene types. Additionally，50 laboratory samples and 113 field samples were tested by this method. Re? sults The established Real?time Fluorescence Quantitative PCR could identify 6 different kdr gene types in A. sinensis. The muta? tion could be detected by single?tube Fluorescence Quantitative PCR，and the detail mutation type could be further identified by double? tube Fluorescence Quantitative PCR. By using this method，50 laboratory samples were confirmed as wild type homozy? gotes. Among 113 field samples，12 were wild type homozygotes，others were L1014F or L1014C mutations，and the total muta? tion frequency was 87.61%. Conclusion The new established TaqMan?MGB Real?time Fluorescence Quantitative PCR can be used to detect the kdr gene L1014 mutations of A. sinensis.

Key words: Anopheles sinensis； kdr gene； Gene mutation；TaqMan?MGB probe； Real?time Fluorescence Quantitative PCR