白纹伊蚊和三带喙库蚊抗药性相关基因突变PCR-RFLP检测方法的建立  

Establishment of a PCR-RFLP method for detecting mutations in genes related to insecticide resistance in Aedes albopictus and Culex tritaeniorhynchus

作　　者：刘鹃[1] 刘鹏 王雅伟 余小梅 邱星辉[2] LIU Juan;LIU Peng;WANG Yawei;YU Xiaomei;QIU Xinghui(Department of Vecor Control,Neijiang Center for Disease Control and Prevention,Neijiang 641100,Sichuan,China;State Key Laboratory of Integrated Managemeat of Pest Insects and Rodents,Institute of Zoology,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]内江市疾病预防控制中心病媒生物防制科,四川内江641100 [2]中国科学院动物研究所,农业虫害鼠害综合治理研究国家重点实验室,北京100101

出　　处：《中国寄生虫学与寄生虫病杂志》2024年第6期744-747,755,共5页Chinese Journal of Parasitology and Parasitic Diseases

摘　　要：目的 基于聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术,建立白纹伊蚊和三带喙库蚊抗药性相关基因突变的快速检测方法。方法 根据白纹伊蚊电压门控钠离子通道(VGSC)和三带喙库蚊VGSC、乙酰胆碱酯酶(AChE)基因序列设计PCR引物,优化酶切位点,建立检测白纹伊蚊和三带喙库蚊抗药性相关基因突变的PCR-RFLP方法。诱蚊灯法采集白纹伊蚊和三带喙库蚊,提取单只蚊虫基因组DNA,以单只蚊虫的基因组DNA为模板进行PCR,分别扩增含1016位点的白纹伊蚊VGSC基因、含1014位点的三带喙库蚊VGSC基因和含455位点的三带喙库蚊AChE基因片段。3种PCR扩增产物分别使用限制性内切酶Bsa JⅠ、DdeⅠ、MboⅠ进行酶切,根据酶切片段的长度多态性来区分个体的基因型。结果 成功建立了检测白纹伊蚊VGSC-V1016G、三带喙库蚊VGSC-L1014F和三带喙库蚊AChE-F455W突变的方法。酶切结果显示,5个白纹伊蚊VGSC扩增产物中有3个野生型1016V纯合子、2个野生型1016V和突变型1016G杂合子,5个三带喙库蚊VGSC扩增产物中有2个野生型1014L纯合子、2个野生型1014L和突变型1014F杂合子、1个突变型1014F纯合子,5个三带喙库蚊AChE扩增产物中有2个野生型455F和突变型455W杂合子、3个突变型455W纯合子。结论 建立的白纹伊蚊和三带喙库蚊抗药性相关基因突变PCR-RFLP检测方法操作方便快捷、成本更低且准确性高。Objective To establish a rapid detection method for detecting mutations in genes related to insecticide resistance in Aedes albopictus and Culex tritaeniorhynchus based on polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)technology.Methods PCR primers were designed based on the gene sequences of voltage-gated sodium channel(VGSC)in Ae.albopictus,VGSC and acetylcholinesterase(AChE)in Cx.tritaeniorhynchus,and the restriction enzyme cutting sites were optimized to establish a PCR-RFLP method for detecting mutations in genes related to insecticide resistance in Ae.albopictus and Cx.tritaeniorhynchus.Mosquitoes were collected by light trap,and genomic DNA was extracted from individual mosquitoes.PCR was performed using genomic DNA from individual mosquitoes as templates to amplify fragments containing the 1016 site of the Ae.albopictus VGSC gene,the 1014 site of the Cx.tritaeniorhynchus VGSC gene and the 455 site of the Cx.tritaeniorhynchus AChE gene.The PCR products of 3 genes were digested with the restriction enzymes BsaJⅠ,DdeⅠand MboⅠ,respectively,and the genotypes of individuals were distinguished based on the length polymorphism of the digested fragments.Results The detection method for the VGSC-V1016G mutation in Ae.albopictus,the VGSC-L1014F mutation and the AChE-F455W mutation in Cx.tritaeniorhynchus was established successfully.The enzyme digestion results showed that among the five Ae.albopictus VGSC amplification products,three were homozygous for the wild-type 1016V,and two were heterozygous for the wild-type 1016V and mutant 1016G.Among the five Cx.tritaeniorhynchus VGSC amplification products,two were homozygous for the wild-type 1014L,two were heterozygous for the wild-type 1014L and mutant 1014F,and one was homozygous for the mutant 1014F.Among the five Cx.tritaeniorhynchus AChE amplification products,two were heterozygous for the wild-type 455F and mutant 455W,and three were homozygous for the mutant 455W.Conclusions The PCR-RFLP method established for detecting mutat

关 键 词：白纹伊蚊 三带喙库蚊 电压门控钠离子通道 乙酰胆碱酯酶 抗性突变 

分 类 号：R384.11[医药卫生—医学寄生虫学]