重组酶介导的斯氏并殖吸虫等温扩增荧光检测方法的建立及检测效果初步评价  

Establishment of a fluorescent recombinase-aided isothermal amplification assay for nucleic acid detection of Paraginiumus skrjabini and preliminary evaluation of its detection efficiency

作　　者：邓艳[1] 刘燕红 陈伟奇[1] 张雅兰[1] 蒋甜甜 李素华[1] 艾琳[3] 蔡茂荣[4] 应清界 刘颖[1] 张红卫[1] DENG Yan;LIU Yan-Hong;CHEN Wei-Qi;ZHANG Ya-Lan;JIANG Tian-Tian;LI Su-Hua;AI Lin;CAI Mao-Rong;YING Qing-Jie;LIU Ying;ZHANG Hong-Wei(Henan Institute of Parasitic Diseases,Henan Center for Disease Control and Prevention,Henan Provincial Key Laboratory for Pathogenic Microorganisms of Infectious Diseases,Zhengzhou 450016;Jiangsu Qitian Gene Technology Co.,Ltd.,China;National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,China;Zhangzhou Center for Disease Control and Prevention,Fujian Province,China)

机构地区：[1]河南省疾病预防控制中心寄生虫病预防控制所、河南省传染病病原生物重点实验室,郑州450016 [2]江苏省奇天生物科技有限公司 [3]中国疾病预防控制中心寄生虫病预防控制所 [4]福建省漳州市疾病预防控制中心

出　　处：《中国血吸虫病防治杂志》2021年第5期464-469,500,共7页Chinese Journal of Schistosomiasis Control

基　　金：河南省科技攻关项目(182102310671)。

摘　　要：目的建立一种基于重组酶介导的等温扩增(recombinase-aided isothermal amplification, RAA)技术的斯氏并殖吸虫快速核酸检测方法,并对其检测效果进行初步评价。方法从溪蟹样本中分离出斯氏并殖吸虫、卫氏并殖吸虫和三平正并殖吸虫囊蚴,提取基因组DNA进行分子鉴定。以斯氏并殖吸虫线粒体细胞色素c氧化酶亚基I基因(cytochrome coxidase 1, cox1)基因序列作为靶序列设计、制备、筛选引物及探针。以河南省济源市和洛阳市宜阳县斯氏并殖吸虫囊蚴基因组DNA为模板进行荧光RAA检测方法验证。以含斯氏并殖吸虫cox1基因序列的不同浓度重组质粒和斯氏并殖吸虫囊蚴基因组DNA为模板进行荧光RAA扩增,评价其检测灵敏度;应用建立的荧光RAA法同时检测卫氏并殖吸虫、三平正并殖吸虫、华支睾吸虫和日本血吸虫基因组DNA,评价其检测特异性。结果从溪蟹样本中分离出斯氏并殖吸虫、卫氏并殖吸虫和三平正并殖吸虫囊蚴,经分子鉴定和系统进化分析确认其与Gen Bank中并殖吸虫标准株基因序列具有同源性。成功建立了斯氏并殖吸虫荧光RAA检测方法,可在5 min内扩增到河南省济源市和洛阳市宜阳县采集的斯氏并殖吸虫囊蚴基因组DNA,而阴性对照无扩增。以重组质粒为模板,荧光RAA法最低检出限为10拷贝/μL重组质粒,且均在5 min内出现阳性扩增;以基因组DNA为模板,可检测到的最低模板DNA浓度为10 pg/μL,且均在10~15 min内出现阳性扩增。荧光RAA法检测卫氏并殖吸虫、三平正并殖吸虫、日本血吸虫和华支睾吸虫基因组DNA结果均为阴性。结论成功建立了一种基于荧光RAA技术的快速、敏感、特异的斯氏并殖吸虫核酸检测方法,在斯氏并殖吸虫病流行区溪蟹现场快速检测与虫种鉴定中具有潜在应用价值。Objective To establish a nucleic acid assay for detection of Paragonimus skrjabini based on the recombinase-aided isothermal amplification(RAA) technique, and to preliminarily evaluate its detection efficiency.Methods The metacercariae of P. skrjabini, P. westermani and Euparagonimus cenocopiosus were isolated from crabs, and genomic DNA was extracted for molecular characterization. The cytochrome c oxidase 1(cox1) gene sequence of P. skrjabini was selected as the target gene fragment, and the primers and probes were designed, screened and synthesized for RAA assay. The genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province were used as templates for verification of the fluorescent RAA assay. The fluorescent RAA assay was performed to detect different concentrations of plasmids containing target gene fragment and P. skrjabini metacercariae genomic DNA to determine the sensitivity. Fluorescent RAA assay was performed with recombinant plasmids containing P. skrjabini cox1 gene sequences at different concentrations and P. skrjabini genomic DNA as templates to evaluate its sensitivity, and the genomic DNA of P. westermani, E. cenocopiosus, Clonorchis sinensis and Schistosoma japonicum was detected with fluorescent RAA assay to evaluate its specificity.Results P. skrjabini, P. westermani and E. cenocopiosus metacercariae were isolated from crabs, respectively. Molecular characterization and phylogenetic analysis confirmed their homology with the genes sequences of standard Paragonimus strains in GenBank. A fluorescent RAA assay was successfully established for nucleic acid detection of P. skrjabini, and the genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province was amplified using the fluorescent RAA assay within 5 min, while the negative control was not amplified. If the recombinant plasmid containing P. skrjabini cox1 gene sequences was used as templates, the fluorescent RAA assay showed the lowest detection l

关 键 词：斯氏并殖吸虫 重组酶介导的等温扩增 核酸检测 检测效果 

分 类 号：R383.23[医药卫生—医学寄生虫学]