Bai Liang, Zhu Guo-Ding, Tang Jian-Xia, Zhang Chao, Liu Yao-Bao, Li Ju-Lin, Cao Jun, Gao Qi
Jiangsu Institute of Parasitic, Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Wuxi 214064, China.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2013 Apr;25(2):167-71, 176.
To establish a Real-time Fluorescence Quantitative PCR to detect the kdr gene mutation in Anopheles sinensis.
One pair of primers and three TaqMan-MGB probes were designed based on kdr gene and its L1014 locus mutations 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.
The established Real-time Fluorescence Quantitative PCR could identify 6 different kdr gene types in A. sinensis. The mutation 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 homozygotes. Among 113 field samples, 12 were wild type homozygotes, others were L1014F or L1014C mutations, and the total mutation frequency was 87.61%.
The new established TaqMan-MGB Real-time Fluorescence Quantitative PCR can be used to detect the kdr gene L1014 mutations of A. sinensis.
建立一种实时荧光定量PCR方法以检测中华按蚊的kdr基因突变。
根据中华按蚊kdr基因及其L1014位点突变设计一对引物和三种TaqMan-MGB探针。优化后，利用6种不同kdr基因类型的中华按蚊样本对实时荧光定量PCR进行验证。此外，用该方法检测了50份实验室样本和113份野外样本。
所建立的实时荧光定量PCR能够鉴定中华按蚊6种不同的kdr基因类型。单管荧光定量PCR可检测到突变，双管荧光定量PCR可进一步鉴定具体突变类型。用该方法检测，50份实验室样本被确认为野生型纯合子。113份野外样本中，12份为野生型纯合子，其他样本为L1014F或L1014C突变，总突变频率为87.61%。
新建立的TaqMan-MGB实时荧光定量PCR可用于检测中华按蚊kdr基因L1014突变。
