应用PCR技术检测中华按蚊蚊胃血血源
Identification of Anopheles sinensis blood meals with PCR
朱长强1** 韩 坤2 艾乐乐1 墙世贵1 韩招久1 姜志宽1 谭伟龙1***
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DOI：10.7679/j.issn.2095-1353.2015.108
作者单位：1.南京军区疾病预防控制中心，南京 210002；2.宁夏回族自治区疾病预防控制中心，银川 750004
中文关键词：中华按蚊，蚊胃血，PCR，吸血习性
英文关键词：Anopheles sinensis, mosquitoes blood meals, PCR, blood-sucking habit
中文摘要：
【目的】 应用PCR技术检测甘肃地区中华按蚊Anopheles sinensis蚊胃血来源，研究其吸血习性。【方法】根据中华按蚊可能吸血对象(人、牛、猪、羊、鼠、鸡)的线粒体DNA细胞色素b序列的差异，设计种特异引物，建立聚合酶链反应（PCR）体系鉴定中华按蚊蚊胃血来源。同时，对人血、猪血、牛血、羊血、鼠血、鸡血及未吸血中华按蚊中提取的DNA进行检测，验证该方法的特异性；对吸饲人血后不同时间（6、12、24、36、48、60 h）的中华按蚊进行检测，测试该方法的敏感性。【结果】 该方法可从已知动物血样和中华按蚊提取的DNA中分别扩增得到689 bp（人）、271 bp（牛）、453 bp（猪）、225 bp（羊）、485 bp（鼠）、266 bp(鸡)和468 bp（中华按蚊）大小的特异性条带；吸人血36 h内的中华按蚊均能扩增出特异性条带，在吸人血后48 h、60 h的中华按蚊中，均未扩出特异性条带。共检测10只现场中华按蚊，血源来自人、牛、猪分别为5只、2只、3只，其中1只蚊子兼吸人血和猪血。【结论】 建立的PCR检测方法可鉴定中华按蚊蚊胃血来源，结果稳定可靠；甘肃地区中华按蚊不仅嗜吸人血，也可兼吸其他动物血。
英文摘要：[Objectives] To develop a PCR method for identifying the source of Anopheles sinensis blood meals and determining the host preferences of A.sinensis. [Methods] Species-specific primers were designed based on the mitochondrial cytochrome b sequences of common mosquito hosts (human, cow, pig, goat, mouse and chicken) to identify the animal blood ingested by wild-caught A. sinensis. Meanwhile, DNA was extracted from the blood of common mosquito hosts, and from A.sinensis that had not ingested a blood meal, to verify the specificity of the method. DNA extracted from A. sinensis mosquitoes 6, 12, 24, 36, 4 h and 60 h after ingesting a blood meal was analyzed to determine the sensitivity of the method. [Results] Specific PCR products (689, 453, 225, 485, 266 and 468 bp) were amplified from the DNA extracted from different animal hosts. Host DNA was detectable in frozen mosquitoes for as long as 36 h after ingestion but could not be amplified 48 h and 60 h after ingestion. PCR analysis of the DNA in wild-caught specimens indicates that A. sinensis fed mainly on the blood of humans (50%), followed by that of pigs (30%) and cows (20%). A mixed blood meal comprised of both human and pig blood was detected in a single mosquito. [Conclusion] The PCR assay proved an accurate and reliable method of identifying the blood meals of wild-caught A. sinensis. A. sinensis feeds on both human and animal blood.