摘要
本研究旨在建立一种基于荧光RPA技术的伊蚊鉴定方法。以白纹伊蚊和埃及伊蚊为例,分别根据其rDNA-ITS2序列设计了特异性RPA引物和探针,通过基础RPA方法进行引物筛选,扩增产物测序后在NCBI数据库中进行Blast比对分析,并与COI基因序列分析结果比较,以验证该方法的准确性。然后,进行特异性、灵敏度、重复性实验。结果显示,本研究建立的荧光RPA方法的鉴定结果与基于COⅠ基因序列的分析鉴定结果一致。该方法在39℃条件下20 min即可完成检测,与刺扰伊蚊、背点伊蚊、三带喙库蚊、中华按蚊、常型曼蚊DNA均无交叉反应,对目的DNA的检测下限为0.01 ng/μL,3个平行的变异系数值均<5%。结果表明,本研究建立的伊蚊荧光RPA鉴定方法,特异性强、灵敏度高、重复性好,操作简便、快速,适合于蚊虫DNA的分子分类鉴别检测。
The objective of this research was to establish a method for identification of Aedes albopictus mosquitoes based on fluorescent RPA.For Ae.albopictus and Aedes aegypti mosquito,the specific RPA primers and probes were designed respectively based on their rDNA-ITS2 sequences.The primers were screened using the basic RPA method.To verify the accuracy of the method,the sequences of RPA product were Blast in NCBI,and then were compared with the result of identification with COI gene.Then,the specificity,sensitivity and repeatability of the method were tested respectively.The results showed that the identification result of the fluorescent RPA method were consistent with the result of the method based on COI gene.The identification of Aedes mosquitoes can be completed at 39℃for 20 min by the fluorescence RPA without cross reactions with the DNA of Ae.vexans,Ae.dorsalis,Culex tritaeniorhynchus,Anopheles sinensis and Mansonia uniformis.The detection limit for the target DNA was 0.01 ng/μL,and the coefficient of variation of three parallel test were all below 5%.The results suggested that the specificity,repeatability,and sensitivity were well,so fluorescent RPA identification of Aedes mosquitoes can be used in molecular detection of mosquitoes.
作者
李颖
张晓龙
慈颖
刘莹莹
杨燕
曹晓梅
LI Ying;ZHANG Xiao-Long;CI Ying;LIU Ying-Ying;YANG Yan;CAO Xiao-Mei(Chinese Academy of Inspection and Quarantine,Beijing 100176,China)
出处
《寄生虫与医学昆虫学报》
CAS
2020年第4期244-250,共7页
Acta Parasitologica et Medica Entomologica Sinica
基金
十三五国家重点研发计划(编号:2018YFF0214903)
十三五科技重大专项(编号:2017ZX10303404-001-004)。
