PCRRFLP单酶切法鉴别赫坎按蚊复合体的研究

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。

Molecular identification of Anopheles hyrcanus complex by using single enzyme digestion PCR-RFLP method

Objective To establish a novel molecular identification method for discrimination of members within Anopheles hyrcanus complex.Methods The sequences of the ribosomal DNA second internal transcribed spacer(rDNA ITS2) region of An.hyrcanus complex,including An.anthropophagus,An.lesteri,An.sinesis and An.yatsushiroensisi were analyzed by using molecular biology software Vector NTI 9.0,and a specificity restriction enzyme was selected based on the restriction fragment length polymorphism.Thus the single enzyme digestion PCR-RFLP method was established for genetic identification of An.hyrcanus complex,and 452 anopheline mosquitoes captured in the field were tested,comparing with the results of the previously established double enzyme digestion PCR-RFLP method and traditional morphological classification.Results The molecular software analysis revealed that the restriction enzyme Dde I could digest rDNA ITS2 region of An.hyrcanus complex into different fragments,thus it could be used for single enzyme PCR-RFLP for An.hyrcanus complex identification,and the result was further confirmed by laboratory experiment.Furthermore,a total of 452 anopheline mosquitoes captured from 4 malaria endemic areas were tested by this single enzyme digestion PCR-RFLP method,and 20 of them were identified as An.anthropophagus,6 as An.lesteri,391 as An.sinesis,and 35 as An.yatsushiroensisi.The results were 100% accordant to the double enzyme digestion PCR-RFLP method,and 93.4% accordant to the traditional morphological classification.Conclusions The newly established single enzyme digestion PCR-RFLP method can be used for An.hyrcanus complex identification,and is more simple and reliable than the traditional morphological classification,and it is a suitable tool for field entomology surveillance.