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

摘要：

目的 目的 建立一种用于中华按蚊杀虫剂抗性相关kdr基因突变检测的实时荧光定量PCR方法。方法 方法 根据中华按蚊kdr基因序列及其L1014位点常见的突变类型设计一对引物和三条TaqMan?MGB探针， 对TaqMan?MGB探针实时荧光定量PCR反应体系和条件进行了优化， 选择经测序鉴定后的6种中华按蚊kdr基因常见类型对该方法进行验证， 并用该方法对50个实验室和113个现场中华按蚊样本进行检测。结果 结果 实时荧光定量PCR方法能对中华按蚊kdr基因6种不同的基因型进行检测， 单管法可判断kdr基因L1014是否发生突变， 双管法可对具体突变类型进一步鉴别。经检测， 50个实验室样本均为野生型纯合体， 而113个现场样本中， 仅12个样本为野生型纯合体， 其余101个样本均发生了L1014F或L1014C 突变， 突变频率为87.61%。结论 结论 TaqMan?MGB探针实时荧光定量PCR可用于中华按蚊kdr基因L1014位点突变的检测。

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