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目的建立一种快速、准确、可视化、易开展的B、E型腺病毒检测技术。方法针对B、E型腺病毒壳蛋白保守序列设计通用引物,建立可同时检测出这两种腺病毒型别的重组酶聚合酶扩增结合侧流层析试纸条(recombinase polymerase amplification combined with a lateral flow dipstick,LFD-RPA)检测技术,评价其灵敏度与特异性,并验证最佳反应温度与反应时间,同时利用该法对19例B、E型腺病毒感染患者及10例健康志愿者鼻咽拭子标本进行检测。结果腺病毒LFD-RPA法检测灵敏度可达10拷贝/μl,与q PCR方法相近,且不会与其他亚属腺病毒及其他病毒发生交叉反应。反应可在25~45℃温度范围内高效进行,检测全程仅需15~20 min。利用临床咽拭子标本对检测体系进行评估,其灵敏度及特异性均达100%。结论该检测方法灵敏度高、特异性强、操作简单,反应快速,能脱离对大型仪器、专业操作人员及正规实验室的依赖,在基层卫生部门,尤其在野外及现场检测中具有极大的应用潜力。
Objective To establish a rapid, accurate, visual, easy to carry out the B, E adenovirus detection technology. Methods A universal primer was designed based on the conserved sequences of the capsid proteins of B and E adenoviruses. A recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) and LFD-RPA. The sensitivity and specificity of this method were evaluated and the optimal reaction temperature and reaction time were validated. Meanwhile, 19 patients with adenovirus type B and E and 10 healthy volunteers Throat swab specimens were tested. Results The sensitivity of LFD-RPA assay was 10 copies / μl, which was close to that of q PCR and did not cross-react with other subgenus adenovirus and other viruses. The reaction can be carried out efficiently in the temperature range of 25-45 ° C, and the whole test only takes 15-20 min. Using clinical throat swab specimens to assess the detection system, the sensitivity and specificity of up to 100%. Conclusion The detection method has high sensitivity, specificity, simple operation, rapid response, and can be relied on for large-scale instruments, professional operators and formal laboratories. It has great application in primary health departments, especially in the field and on-site testing potential.