双探针荧光重组酶聚合酶扩增法检测细粒棘球绦虫技术的建立与应用  

Establishment and application of a dual-probe fluorescent recombinase polymerase amplification assay for detection of Echinococcus granulosus

作　　者：杜建伯 苏雅馨 霍乐乐 王莹 王旭 姜斌 陈雨晴 沈玉娟 DU Jianbo;SU Yaxin;HUO Lele;WANG Ying;WANG Xu;JIANG Bin;CHEN Yuqing;SHEN Yujuan(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Chinese Center for Tropical Diseases Research,National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,Key Laboratory on Parasite and Vector Biology,National Health Commission,WHO Collaborating Centre for Tropical Diseases,National Center for International Research on Tropical Diseases,Ministry of Science and Technology,Shanghai 200025,China)

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心),传染病溯源预警与智能决策全国重点实验室,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,上海200025

出　　处：《中国寄生虫学与寄生虫病杂志》2025年第1期61-68,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(82072307,82372283)。

摘　　要：目的结合重组酶聚合酶扩增技术(RPA),建立一种快速、便捷的检测棘球绦虫及进一步鉴定细粒棘球绦虫的方法。方法以棘球绦虫细胞色素C氧化酶亚基1(co1)和NADH脱氢酶亚基5(nd5)的基因为靶序列,利用Primer Premier 6软件设计特异性引物和探针并进行筛选,建立棘球绦虫核酸双探针荧光RPA检测方法。采用不同DNA拷贝数(10^(4)、10^(3)、10^(2)、10、1个拷贝/µl)的含co1和nd5的重组质粒,评价该方法的敏感度。以多房棘球绦虫、猪带绦虫、日本血吸虫、美洲钩虫、巴西日本圆线虫、华支睾吸虫、蓝氏贾第鞭毛虫、微小隐孢子虫、刚地弓形虫和田鼠巴贝虫基因组DNA评价该方法的特异性。利用23份模拟样品(棘球绦虫原头节DNA和阴性犬粪提取的DNA混合)和5份现场采集的犬粪细粒棘球绦虫阳性DNA样品(经PCR鉴定)评价该方法的可行性。结果针对棘球绦虫基因组DNA建立的FAM/HEX双探针荧光RPA检测法的最佳反应条件为39℃、金属浴300 rpm振荡反应4 min后,采用荧光定量PCR(qPCR)检测荧光信号12.5 min,总反应时长16.5 min。敏感度检测结果显示,该法对co1和nd5重组质粒的最低检出限分别为10拷贝/µl和100拷贝/µl。特异性检测结果显示,该法仅对细粒棘球绦虫有FAM/HEX双重荧光信号,对多房棘球绦虫有FAM单荧光信号,而对猪带绦虫、日本血吸虫、美洲钩虫和华支睾吸虫等9种寄生虫基因组DNA无特异性反应。可行性评价结果显示,含有细粒棘球绦虫基因组DNA的16份模拟样品产生FAM/HEX双阳性信号,其中8份为混合细粒棘球绦虫基因组DNA的模拟样品,8份为混合细粒和多房棘球绦虫基因组DNA的模拟样品,仅含有多房棘球绦虫基因组DNA的7份模拟样品产生FAM单阳性信号,5份现场采集的犬粪细粒棘球绦虫阳性DNA样品产生FAM/HEX双阳性信号,其他DNA样品均无任何阳性信号。该法的检测结果与PCR法的检测结果一致。结论本�Objective To establish a rapid and convenient assay for detection of Echinococcus and identifica-tion of E.granulosus based on recombinant polymerase amplification(RPA).Methods The Echinococcus cytochrome C oxidase subunit 1(co1)and NADH dehydrogenase subunit 5(nd5)of genes were selected as target sequences,and specific primers and probes were designed with the software Primer Premier 6 and screened to establish a dual-probe fluorescent RPA assay for detection of Echinococcus nucleic acid.The sensitivity of the dual-probe fluorescent RPA assay was evaluated with co1 and nd5 recombinant plasmid DNA at concentrations of 10^(4)、10^(3)、10^(2),10,and 1 copies/µl,and the specificity of the assay was evaluated with genomic DNA from E.multilocularis,Taenia solium,Schistosoma japonicum,Necator americanus,Nippostrongylus brasiliensis,Clonorchis sinensis,Giardia lamblia,Cryptosporidium parvum,Toxoplasma gondii and Babesia microti.The feasibility of the method was evaluated using 23 simulated samples(pre-pared by mixing E.granulosus protoscolex DNA with DNA extracted from negative canine feces)and 5 field-collected dog fecal samples positive for E.granulosus(confirmed by PCR).Results The optimal reaction procedure of the FAM/HEX dual-probe fluorescence RPA assay for detection of Echinococcus genomic DNA was oscillation in a metal bath at 39℃,300 r/min for 4 minutes,followed by detection of the fluorescence signal for 12.5 minutes using real-time quantitative PCR(qPCR)assay.The total reaction time of performance was 16.5 minutes.The minimum detection limits of the dual-probe fluorescent RPA assay were 10 copies/µl for the co1 recombinant plasmid and 100 copies/µl for the nd5 recombinant plasmid,respectively,and the assay yielded the FAM/HEX dual fluorescence signals for E.granulosus,FAM single fluorescence signal for E.multilocularis,and no specific reaction to the genomic DNA from T.solium,S.ja⁃ponicum,N.americanus,N.brasiliensis,C.sinensis,G.lamblia,C.parvum,T.gondii and B.microti.In simulated DNA samples,FAM/HEX

关 键 词：棘球绦虫 细粒棘球绦虫 重组酶聚合酶扩增 快速鉴别 

分 类 号：R383.33[医药卫生—医学寄生虫学]