猫源蓝氏贾第虫HRM快速基因分型方法的建立  

Development of a rapid high resolution melting genotyping method for cat-derived Giardialamblia

作　　者：谭立娉[1] 郑国超 李雅文[1] 胡伟[1] 罗琴[1] 林丽琴[1] 路鹏云[1] 余新刚[1] 武省[1] 宋美冉 王祯[1] 江飚[1] 李国清[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医科学》2015年第6期602-608,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31272551)

摘　　要：为建立一种蓝氏贾第虫的快速基因分型方法,选取β-贾第素(β-giardin)基因作为遗传标志,采用HRM技术对华南地区常见的猫源蓝氏贾第虫A型、F型进行基因分型。根据GenBank中登录的序列KJ027416.1(A型)和JX 275388(F型)设计了qPCR-HRM引物BGF和BGR,通过对反应温度和反应体系进行优化,建立了HRM基因分型方法,并对其稳定性、敏感性和准确性进行了检测。结果显示,HRM分型方法可对蓝氏贾第虫A型、F型以及混合感染情况进行检测;批内和批间重复性均良好;当样品质量浓度低至4.27×10-4 ng/μL时,HRM检测方法依然可以分辨出基因型;其准确性与测序结果完全一致。结果表明,建立的HRM基因分型方法具有快速、稳定、敏感和准确的特点,适用于猫源蓝氏贾第虫的临床检测和贾第虫病的分子流行病学调查。To establish a rapid method for genotyping of Giardia lamblia,the common cat-derived G.lambliaassemblages A and F in South China were genotyped by high resolution melting(HRM)technology,according toβ-giardin gene sequence.Based on the sequence KJ 027416.1(assemblage A)and JX 275388(assemblage F)in GenBank,qPCR-HRM primers BGF and BGR were designed.Its reaction temperature and reaction system were optimized,the stability,sensitivity and accuracy of the HRM method were tested.Results showed that the HRM genotyping method could detect successfully assemblages of A,F and A&F of G.lamblia.Intra-and inter-assay reproducibility was good,with the lowest detection concentration of4.27×10-4 ng/μL sample.Its accuracy was in complete accord with sequencing results.In conclusion,the HRM genotyping method is rapid,stable,specific,highly sensitive,and suitable for clinical detection of catderived G.lambliaand molecular epidemiological survey of giardiasis.

关 键 词：蓝氏贾第虫 HRM 基因分型 

分 类 号：S858.293[农业科学—临床兽医学]