收稿日期: 2020-01-02
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)
Establishment of recombinase polymerase amplification technique for rapid detection of Schistosoma japonicum nucleic acid
Received date: 2020-01-02
Online published: 2020-07-07
Supported by
National Science and Technology Major Project(2018ZX10101002-002);National Science and Technology Major Project(2018ZX10004222-004)
目的 应用重组酶聚合酶扩增（RPA）技术,建立一种敏感、特异、简便且快速的日本血吸虫核酸检测方法。 方法 选择Sj28S核糖体基因片段为靶序列,用Primer Premier 5软件设计特异性引物,建立RPA扩增反应,并进行优化以确定最佳反应条件。提取不同虫期日本血吸虫,以及曼氏血吸虫、埃及血吸虫、单尾尾蚴感染钉螺、华支睾吸虫、大片形吸虫、卫氏并殖吸虫和牛带绦虫等基因组DNA,评价所建立方法的特异性和敏感性,并对不同混合比例（1 : 10、1 : 50、1 : 100、1 : 250、1 : 500、1 : 1 000、1 : 2 000）的阳性和阴性钉螺基因组DNA的检出性能和重复性进行评价。 结果 建立的RPA方法可特异地扩增出日本血吸虫216 bp大小的目的基因片段。RPA的最佳反应条件为39 ℃、20 min。该方法与曼氏血吸虫、埃及血吸虫、单尾尾蚴感染钉螺、华支睾吸虫、大片形吸虫、卫氏并殖吸虫和牛带绦虫及阴性钉螺基因组DNA均无交叉反应。针对日本血吸虫成虫基因组的最低检出限为100 fg/μl,针对重组质粒的最低检出限为100拷贝/μl,且血吸虫不同虫期的基因组DNA样本均能被准确检出。应用建立的RPA方法检测不同混合比例钉螺DNA混合样品,结果显示,最低检出比例为1 : 1 000。重复性试验结果显示,5次检测结果完全一致,无假阴性和假阳性。 结论 建立了日本血吸虫RPA检测方法,该法敏感性高、特异性好、简便快速,有望用于血吸虫感染的快速检测及风险监测。
王盛琳 , 邓王平 , 李银龙 , 王丽萍 , 张利娟 , 吕山 , 许静 . 重组酶聚合酶扩增技术快速检测日本血吸虫核酸方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2020 , 38(3) : 293 -298 . DOI: 10.12140/j.issn.1000-7423.2020.03.006
Objective To establish a sensitive, specific, simple and rapid method for detecting Schistosoma japonicum nucleic acid by using the recombinase polymerase amplification (RPA) technique. Methods Specific primers were designed to target the 28S ribosomal S. japonicum (Sj28S) gene using Primer Premier 5 software. The RPA method was established and the reaction conditions were optimized. The specificity and sensitivity of the method were tested using genomic DNA extracted from S. japonicum at different stages, S. mansoni, S. haematobium, Oncomelania hupensis infected with single-tail cercariae, Clonorchis sinensis, Fasciola gigantica, Paragonimus westermani, and Taenia saginata. The detection performance and reproducibility of the RPA method were assessed using the mixed genomic DNA from infected and non-infected O. hupensis at various ratios (1 : 10, 1 : 50, 1 : 100, 1 : 250, 1 : 500, 1 : 1 000, 1 : 2 000). Results The RPA method specifically amplified the 216-bp target gene fragment of S. japonicum. The optimal reaction conditions of RPA were at 39 ℃ for 20 min. No cross reactions occurred with the genomic DNA of S. mansoni, S. haematobium, O. hupensis infected with single-tail cercariae, C. sinensis, F. gigantica, P. westermani, T. saginata and O. hupensis with negative infection. The lowest detectable limit of the RPA method for genomic DNA of S. japonicum adults was 100 fg/μl, and for the recombinant plasmid was 100 copies/μl. In addition, the RPA method could exactly detected the target DNA of S. japonicum at different stages. Tested with mixed genomic DNA of infected and all snails at various ratios, the lowest detectable limit of the RPA method for the target DNA fragment was 1 : 1 000. The test was repeated for 5 times, and all produced consistent results, with no false negative or false positive results. Conclusion The RPA method established for detecting S. japonicum nucleic acid displays high sensitivity and specificity, and is simple and rapid to use. Thus, the method could be used for rapid detection and risk monitoring.
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