TaqManMGB探针实时荧光定量PCR用于中华按蚊kdr基因突变检测的研究

Study on TaqMan-MGB Real-time Fluorescence Quantitative PCR to detect gene mutation of kdr from Anopheles sinensis

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。

Objective To establish a Realtime Fluorescence Quantitative PCR to detect the kdr gene mutation in Anopheles sinensis. Methods One pair of primers and three TaqManMGB probes were designed based on kdr gene and its LlO14 locus mu tations ofA. sinensis. After optimization, the Realtime Fluorescence Quantitative PCR was verified by using 6 types ofA. sinensis samples with different kdr gene types. Additionally, 50 laboratory samples and 113 field samples were tested by this method. Re sults The established Realtime Fluorescence Quantitative PCR could identify 6 different kdr gene types in A. sinensis. The muta tion could be detected by singletube Fluorescence Quantitative PCR, and the detail mutation type could be further identified by doubletube Fluorescence Quantitative PCR. By using this method, 50 laboratory samples were confirmed as wild type homozy gores. Among 113 field samples, 12 were wild type homozygotes, others were L IOldF or L1014C mutations, and the total muta tion frequency was 87.61%. Conclusion The new established TaqManMGB Realtime Fluorescence Quantitative PCR can be used to detect the kdr gene L1014 mutations ofA. sinensis.