qPCR检测白纹伊蚊携带Wolbachia分型及密度方法的建立与应用  

ESTABLISHMENT AND APPLICATION OF A QPCR-BASED METHOD FOR WOLBACHIA GENOTYPE AND DENSITY DETECTION IN AEDES ALBOPICTUS

作　　者：熊易平 周宁馨 梁国锐 李建航 李博 韩宛蓉 邢丹 郭晓霞 姜玉庭 赵彤言 XIONG Yi-ping;ZHOU Ning-xin;LIANG Guo-rui;LI Jian-hang;LI Bo;HAN Wan-rong;XING Dan;GUO Xiao-xia;JIANG Yu-ting;ZHAO Tong-yan(Beijing Key Laboratory of Vector Borne and Natural Focus Infectious Diseases,State Key Laboratory of Pathogen and Biosecurity,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China;Public Health School,Fujian Medical University,Fuzhou 350122,Fujian,China;Artemisinin Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China;School of Public Health,the Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang 550025,Guizhou,China;Life Science College,Southwest Forestry University,Kunming 650224,Yunnan,China)

机构地区：[1]军事科学院军事医学研究院,病原微生物生物安全全国重点实验室,媒介生物危害和自然疫源性疾病北京市重点实验室,北京100071 [2]福建医科大学公共卫生学院,福州350122 [3]广州中医药大学青蒿研究中心,广州510405 [4]贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵阳550025 [5]西南林业大学生命科学学院,昆明650000

出　　处：《寄生虫与医学昆虫学报》2024年第1期19-27,共9页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家科技部传染病重大专项(2017ZX0303404)。

摘　　要：目的白纹伊蚊天然携带A与B亚组沃尔巴克氏体Wolbachia,这种共生菌能够通过抑制宿主传播病毒能力或降低宿主种群密度来控制虫媒疾病传播,且这种作用强度与其密度密切相关,本文旨在建立一种准确检测蚊虫携带Wolbachia分型与密度的方法。方法克隆白纹伊蚊携带Wolbachia的wsp基因并测序,在w AlbA与w AlbB wsp基因的非保守区设计特异性引物,建立qPCR检测方法。结果该方法灵敏性和特异性良好,两亚组Wolbachia无交叉反应。利用该方法检测不同浓度四环素处理后蚊虫携带Wolbachia密度下降程度,确定最适四环素处理浓度为0.1 mg/mL。结论本方法的建立为研究Wolbachia在蚊虫生理生殖和天然免疫方面的作用提供了技术支撑。Objective Naturally,Aedes albopictus carries both the A and B supergroups of Wolbachia strains,which are symbiotic bacteria that can influence the transmission of mosquito-borne diseases by inhibiting the ability of their host to transmit viruses or by reducing the population density of their host,and the strength of this effect is closely related to its density in host cells.In this study,an accurate qPCR-based method for detecting Wolbachia genotype and density in mosquitoes was established.Methods The wsp gene of Wolbachia carried by Ae.albopictus was cloned and sequenced,and specific primers for the non-conserved regions of the w AlbA and w AlbB wsp genes were designed.Results The application of this method showed great sensitivity and specificity.The primers did not cross-react between the two Wolbachia supergroups.Further,with this method,it was possible to detect decreases in Wolbachia density in host cells following treatment with tetracycline at different concentrations,and the optimal tetracycline treatment concentration was determined to be 0.1 mg/mL.Conclusion This method can provide technical supports for studies of the effects of Wolbachia on physiological reproduction and innate immunity in mosquitoes.

关 键 词：白纹伊蚊 沃尔巴克氏体 qPCR 四环素 

分 类 号：R384.1[医药卫生—医学寄生虫学]