中华按蚊和雷氏按蚊实时荧光定量PCR鉴定方法的建立与应用
Establishment of real-time PCR methods to identify Anopheles sinensis and Anopheles lesteri
舒黄芳，王可艺，吴 德，林荣幸，卢文成，阮彩文，邓卓晖，张 萌，宋 铁
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DOI：10.7679/j.issn.2095-1353.2023.033
作者单位：广东省疾病预防控制中心，广州 511430
中文关键词：中华按蚊；雷氏按蚊；TaqMan探针；实时荧光定量PCR
英文关键词：Anopheles sinensis; Anopheles lesteri; TaqMan probe; Real-time fluorescence quantitative PCR
中文摘要：
【目的】 建立中华按蚊Anopheles sinensis与雷氏按蚊Anopheles lesteri实时荧光定量PCR（qPCR）检测技术，为2种传疟按蚊的准确鉴定提供分子鉴定方法。【方法】 建立2种按蚊的样本库，利用形态学方法和基因测序对蚊虫样本进行鉴定；合成并克隆阳性质粒质控品。从NCBI数据库上下载中华按蚊和雷氏按蚊的rDNA-ITS2序列，利用Prime 3软件在线设计2种按蚊的引物和探针，并验证该PCR方法的最低检测限、敏感性、特异性和可重复性。【结果】 收集并鉴定4种383头蚊虫，其中中华按蚊159头、雷氏按蚊104头、致倦库蚊Culex pipiens quinquefasciatus 60头和白纹伊蚊Aedes albopictus 60头。中华按蚊和雷氏按蚊的qPCR的最低检测限均为31.50 拷贝/反应，标准曲线线性关系相关系数（R2）均为0.99。敏感性和特异性均为100%，重复性试验结果显示，中华按蚊和雷氏按蚊的组内和组间变异系数均小于2%。【结论】 本方法所建立的TaqMan探针qPCR方法灵敏度高，特异性高，可用于中华按蚊和雷氏按蚊的分子鉴别。
英文摘要：
[Objectives] To develop a real-time PCR technique to identify Anopheles sinensis and Anopheles lesteri. [Methods] Samples of each species were obtained and identified using both morphological methods and gene sequencing. Positive plasmid quality control products were synthesized and cloned, and the rDNA-ITS2 sequences of A. sinensis and A. lesteri were downloaded from the NCBI database. Prime 3 software was used to design primers and probes for each species online, and the minimum detection limit, sensitivity, specificity and repeatability of the PCR method were verified. [Results] Three hundred and eighty-three mosquitoes were collected and identified to 4 species; 159 A. sinensis, 104 A. lesteri, 60 Culex pipiens quinquefasciatus and 60 Aedes albopictus. The minimum detection limit of qPCR for A. sinensis and A. lesteri was 31.5 copy/response and the linear correlation coefficient (R2) of the standard curve was 0.99. The results of a repeatability test showed that intra and inter-group coefficients of variation for A. sinensis and A. lesteri were < 2%. [Conclusion] A TaqMan probe, real-time, fluorescent, quantitative PCR method with good sensitivity and specificity was developed to identify A. sinensis and A. lesteri.