基于重组酶聚合酶扩增结合侧流层析技术的B、E型腺病毒快速可视化检测方法的研究与评价

Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of adenovirus

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 develop a rapid,accurate,visual,and portable detection method for adenovirus types B（ Adv B） and E（ Adv E）. Methods Universal primers were targeted on type-specific conserved regions to allow the simultaneous detection of both human Adv（ HAd V） species. A detection method based on the combination of recombinase polymerase amplification（ RPA） and lateral flow dipstick（ LFD） was established the sensitivity and specificity evaluated,and throat swab specimens of 19 patients infected with Adv B and Adv E as well as 10 healthy volunteers were detected with this method. Results The detection limit of the method was 10 copies/μl Adv DNA,which was close to that of q PCR,and there were no cross-reactions with other species of Adv and unrelated virus. The detection could be finished within 15 to 20 min within the temperature range of 25 to 45℃. When applied to clinical samples,this method showed 100% sensitivity and specificity. Conclusion This detection assay is a sensitive,specific,rapid and simple method that eliminates the need for expensive equipment,trained personnel or laboratories. The characteristics of this system render it suitable for use in grass-roots healthcare departments,and the system is especially effective for field testing and on-site testing.