摘要
【目的】建立中华按蚊Anopheles sinensis与雷氏按蚊Anopheles lesteri实时荧光定量PCR(qPCR)检测技术,为2种传疟按蚊的准确鉴定提供分子鉴定方法。【方法】建立2种按蚊的样本库,利用形态学方法和基因测序对蚊虫样本进行鉴定;合成并克隆阳性质粒质控品。从NCBI数据库上下载中华按蚊和雷氏按蚊的rDNA-ITS2序列,利用Prime3软件在线设计2种按蚊的引物和探针,并验证该PCR方法的最低检测限、敏感性、特异性和可重复性。【结果】收集并鉴定4种383头蚊虫,其中中华按蚊159头、雷氏按蚊104头、致倦库蚊Culex pipiens quinquefasciatus 60头和白纹伊蚊Aedes albopictus 60头。中华按蚊和雷氏按蚊的qPCR的最低检测限均为31.50拷贝/反应,标准曲线线性关系相关系数(R^(2))均为0.99。敏感性和特异性均为100%,重复性试验结果显示,中华按蚊和雷氏按蚊的组内和组间变异系数均小于2%。【结论】本方法所建立的TaqMan探针qPCR方法灵敏度高,特异性高,可用于中华按蚊和雷氏按蚊的分子鉴别。
[Objectives]To develop a real-time PCR technique to identify Anopheles sinensis and Anopheles lesteri.[Methods]Samples of each species were obtained and identified using both morphological methods and gene sequencing.Positive plasmid quality control products were synthesized and cloned,and the rDNA-ITS2 sequences of A.sinensis and A.lesteri were downloaded from the NCBI database.Prime 3 software was used to design primers and probes for each species online,and the minimum detection limit,sensitivity,specificity and repeatability of the PCR method were verified.[Results]Three hundred and eighty-three mosquitoes were collected and identified to 4 species;159 A.sinensis,104 A.lesteri,60 Culex pipiens quinquefasciatus and 60 Aedes albopictus.The minimum detection limit of qPCR for A.sinensis and A.lesteri was 31.5 copy/response and the linear correlation coefficient(R^(2))of the standard curve was 0.99.The results of a repeatability test showed that intra and inter-group coefficients of variation for A.sinensis and A.lesteri were<2%.[Conclusion]A TaqMan probe,real-time,fluorescent,quantitative PCR method with good sensitivity and specificity was developed to identify A.sinensis and A.lesteri.
作者
舒黄芳
王可艺
吴德
林荣幸
卢文成
阮彩文
邓卓晖
张萌
宋铁
SHU Huang-Fang;WANG Ke-Yi;WU De;LIN Rong-Xing;LU Wen-Cheng;RUAN Cai-Wen;DENG Zhuo-Hui;ZHANG Meng;SONG Tie(School of Public Health,Guangdong Pharmaceutical University,Guangzhou 510315,China;Institute for Parasitic Disease Control and Prevention,Guangdong Provincial Center for Disease Control and Prevention,Guangzhou 511430,China;Institute for Communicable Disease Control and Prevention,Guangdong Provincial Center for Disease Control and Prevention,Guangzhou 511430,China;Guangdong Provincial Center for Disease Control and Prevention,Guangzhou 511430,China)
出处
《应用昆虫学报》
CAS
CSCD
北大核心
2023年第1期304-312,共9页
Chinese Journal of Applied Entomology
基金
广东省医学科学技术研究基金(B2020140)
