收稿日期: 2020-03-13
网络出版日期: 2020-07-07
基金资助
国家科技重大专项(2018ZX10101002-002);国家科技重大专项(2018ZX10004222-004);第60批中国博士后科学基金面上资助(2016M600111)
Development and preliminary evaluation of a rapid visualization detection method for circulating nucleic acids of Schistosoma japonicum based on RPA-LFD
Received date: 2020-03-13
Online published: 2020-07-07
Supported by
National Science and Technology Major Project(2018ZX10101002-002);National Science and Technology Major Project(2018ZX10004222-004);the 60th Batch of General Financial Grant from the China Postdoctoral Science Foundation(2016M600111)
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10-7 ng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10-6 ng (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。
邓王平 , 洪清华 , 徐斌 , 王盛琳 , 王丽萍 , 许静 , 胡薇 , 周晓农 . 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[J]. 中国寄生虫学与寄生虫病杂志, 2020 , 38(3) : 286 -292 . DOI: 10.12140/j.issn.1000-7423.2020.03.005
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 fragment in mice dummy positive sera, and the free SjCHGCS19 DNA fragment in the infected mice sera on days 7, 21 and 35 after infection. Conclusion A RPA-LFD method was established for rapid visualizing detection of circulating nucleic acids of S. japonicum. This method is highly sensitive and has potential applicability for early detection of schistosome infection.
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