基于RPA-LFD的日本血吸虫循环核酸快速可视化检测方法的建立及初步评价  

Development and preliminary evaluation of a rapid visualization detection method for circulating nucleic acids of Schistosoma japonicum based on RPA-LFD

作　　者：邓王平 洪清华 徐斌 王盛琳 王丽萍 许静 胡薇 周晓农[1] DENG Wang-ping;HONG Qing-hua;XU Bin;WANG Sheng-lin;WANG Li-ping;XU Jing;HU Wei;ZHOU Xiao-nong(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Chinese Center for Tropical Diseases Research,WHO Collaborating Center of Tropical Diseases,National Center for International Research on Tropical Diseases,Ministry of Science and Technology,Key Laboratory of Parasite and Vector Biologyy Ministry of Healthy Shanghai 200025,China;Department of Microbiology and Microbial Engineeringy School of Life Sciences,Fudan Universityy Shanghai 200433,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,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,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,上海200025 [2]复旦大学生命科学学院微生物学与微生物工程系,上海200433

出　　处：《中国寄生虫学与寄生虫病杂志》2020年第3期286-292,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家科技重大专项(No.2018ZX10101002-002,No.2018ZX10004222-004);第60批中国博士后科学基金面上资助(No.2016M600111)。

摘　　要：目的结合重组酶聚合酶扩增(RPA)技术及侧流层析试纸条法(LFD)建立日本血吸虫特异核酸片段的快速可视化检测方法,并初步评价其检测日本血吸虫感染小鼠血清中血吸虫循环核酸的应用价值。方法以日本血吸虫非长末端重复序列逆转录转座子SjCHGCS19为靶标,设计RPA引物和探针,以日本血吸虫基因组DNA为模板进行RPA及LFD检测,并优化RPA反应温度及时间,建立日本血吸虫核酸RPA-LFD快速可视化检测方法。用RPA-LFD检测模板量为10^-1、10^-2、10^-3、10^-4、10^-5、10^-6和10^-7ng的日本血吸虫基因组DNA,评价其敏感性;用RPA-LFD方法检测日本血吸虫、埃及血吸虫、曼氏血吸虫、卫氏并殖吸虫及华支睾吸虫基因组DNA,评价其特异性。制备含0.01、0.1、1、10和100 ng日本血吸虫成虫基因组DNA的模拟阳性鼠血清。用40条日本血吸虫尾蚴感染小鼠,采集并分离感染前及感染后7、21、35 d小鼠尾静脉血清。提取模拟阳性鼠血清、感染小鼠血清样品中的循环DNA,评价RPA-LFD检测血清中血吸虫特异核酸的可行性及其早期检测价值。结果建立了快速可视化检测SjCHGCS19重复序列的RPA-LFD方法,30~45℃反应10 min即可检出目的片段。RPA最优反应条件为39℃,20 min。RPA-LFD对日本血吸虫成虫基因组DNA的最低检出限为10^-6ng(1 fg)。特异性评价结果显示,RPA-LFD检测曼氏血吸虫和埃及血吸虫基因组DNA结果为阳性,检测卫氏并殖吸虫和华支睾吸虫基因组DNA结果为阴性。RPA-LFD方法可成功检出含0.01~100 ng日本血吸虫基因组DNA的模拟阳性鼠血清以及血吸虫感染后7、21、35 d鼠血清中的游离SjCHGCS19 DNA片段。结论建立了一种快速、可视化检测日本血吸虫循环核酸的RPA-LFD方法,该方法敏感性高,具有检测血吸虫早期感染的潜在应用价值。Objective To develop a rapid visualization method for specific nucleic acid fragments of Schistosoma japonicum by combining recombinase polymerase amplification(RPA)and lateral flow dipstick(LFD)methods,and evaluate its application value in detecting circulating nucleic acid of Schistosoma spp.in infected mouse serum.Methods RPA primers and probe were designed to target the S.japonicum non-long terminal repeat retrotransposons SjCHGCS19.RPA was performed using S.japonicum genomic DNA as the template,and the amplification product was examined by the LFD method.After optimizing the RPA reaction temperature and time,the RPA-LFD assay was established.The sensitivity of RPA-LFD assay was evaluated by detecting 10^-1,10^-2,10^-3,10^-4,10^-5,10^-6 and 10^-7 ng of S.japonicum genomic DNA;and the assay specificity was evaluated by detecting genomic DNA from S.japonicum,S.haematobium,S.mansoni,Paragonimus westermani and Clonorchis sinensis.Mouse dummy positive sera containing 0.01,0.1,1,10 or 100 ng of S.japonicum adult worm genomic DNA were prepared,and mouse experiment was conducted by infection with 40 S.japonicum cerariae,from which tail vein serum was obtained before infection and on days 7,21 and 35 after infection.Then,circulating DNA was extracted from mice dummy positive sera and infected mice sera,and examined by the RPA-LFD method,to evaluate the method’s feasibility and value of early detection in assaying specific schistosome nucleic acids in serum samples.Results The RPA-LFD assay was established to rapidly detect SjCHGCS19 repeat sequence with visualized results.The target fragments could be detected in 10 min,at 30-45℃.The optimal reaction condition was 39℃for 20 min.The lowest detectable limit for S.japonicum adult worm genomic DNA was 10^-6 ng(1 fg).Evaluation of the specificity indicated that the RPA-LFD method showed cross-reaction with the genomic DNA of S.mansoni and S.haematobium,but not with that of P.westermani and C.sinensis.The RPA-LFD assay was able to detect 0.01-100 ng of schistosome DNA

关 键 词：日本血吸虫 重组酶聚合酶扩增 侧流层析试纸条 循环核酸 可视化检测 

分 类 号：R383.24[医药卫生—医学寄生虫学]