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目的建立一种新的赫坎按蚊复合体近缘种按蚊基因鉴别技术,应用于现场按蚊样本的鉴别。方法用分子生物学软件Vector NTI 9.0,对赫坎按蚊复合体的嗜人按蚊、雷氏按蚊、中华按蚊、八代按蚊4个近缘种按蚊rDNA基因的ITS2区段序列进行限制性内切酶酶切位点预测分析,选择特异性内切酶,建立一种能同时鉴别4种近缘种按蚊的聚合酶链反应限制性片段长度多态(PCR RFLP)单酶切法鉴别技术,并对现场捕获的452只按蚊样本进行基因鉴别。结果软件预测赫坎按蚊复合体近缘种4种按蚊的rDNA基因的ITS2区段可被限制性内切酶Dde I切成大小易于区分的不同片段,PCR RFLP单酶切实验结果和软件预测结果完全吻合。4个疟疾流行区捕获的452只按蚊样本经PCR RFLP Dde I单酶切法鉴定有20只嗜人按蚊、6只雷氏按蚊、391只中华按蚊、35只八代按蚊,与形态学鉴别结果符合率为93.4%,与PCR RFLP双酶切法结果符合率为100%。结论新建立的PCR RFLP Dde I单酶切法基因鉴别技术可准确鉴别赫坎按蚊复合体,比传统的形态学鉴别更为简便、可靠,适合用于复合媒介地区的媒介监测。
OBJECTIVE: To establish a new Anopheles stephensi genetic identification technique for Anchorage mosquito in Herkan, which is used in the identification of Anopheles sinensis samples. Methods The molecular biology software Vector NTI 9.0 was used to sequence the ITS2 segment of Anopheles anthropophagus, Anopheles spp., Anopheles sinensis and Anopheles sinensis in four closely related species A restriction endonuclease digestion analysis was carried out to select specific endonucleases and establish a restriction enzyme digestion restriction fragment length polymorphism (PCR RFLP) single digestion France identification technology, and 452 samples of Anopheles mosquitoes captured in the field were identified. Results The software predicted that ITS2 region of rDNA gene of Anopheles sinensis complex was closely segmented by restriction endonuclease Dde I and PCR RFLP single digestion results and software The forecast result is completely consistent. A total of 452 Anopheles sinensis samples collected from 4 malaria endemic areas were identified by PCR RFLP Dde I digestion with 20 Anopheles anthropophagus, 6 Anopheles spp., 391 Anopheles sinensis and 35 Anopheles spp. The coincidence rate of morphological identification was 93.4%, which coincided with the result of PCR RFLP double digestion method was 100%. Conclusion The newly established PCR RFLP Dde I single enzyme digestion method can accurately identify H. hekanum complex, which is more simple and reliable than the traditional morphological identification and is suitable for the media monitoring in the complex media area.