基于颜色判定的RT-LAMP技术检测HCoV-OC43冠状病毒

Visual detection of human coronavirus OC43 by reverse transcription loop-mediated isothermal amplification

目的 建立基于颜色判定的适用于国境口岸简便、快速、灵敏和特异的逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)方法检测人冠状病毒OC43(HCoV-OC43)核酸。方法 通过序列比对,选择HCoV-OC43(Gen Bank号:KU131570)为参考株,根据其核衣壳(nucleocapsid,N)蛋白基因保守区序列设计6条特异性引物,在65℃等温条件下扩增45min。在扩增前加入钙黄绿素作为反应指示剂,以最终反应颜色变化作为结果判断标准。对病毒核酸依次倍比稀释以检验该方法的灵敏性;同时,对甲型、乙型流感病毒和其他HCoV进行检测,以检验该方法的特异性。结果 通过引物筛选最终选定了1组特异性引物,仅能与HCoV-OC43病毒核酸产生特异性反应并出现颜色改变,而与其他病毒均不能产生颜色变化,具有较高的特异性。与常规荧光定量RT-PCR法相比,该方法检测灵敏度达5.6拷贝/反应,灵敏度更高,且在短时间内达到对HCoV-OC43基因快速检测的目的。结论 建立的基于颜色判定的RT-LAMP方法可实现对HCoV-OC43病毒快速、灵敏、特异性检测,具有在国境口岸以及基层医疗机构和疫区现场推广应用的潜力。

Objective To develop a sensitive and simple assay for the rapid detection of human coronavirus OC43（HCoV-OC43） by colorimetic reverse transcription loop-mediated isothermal amplification（RT-LAMP）. Methods The method employed six specially designed primers that recognized eight distinct regions of HCoV-OC43（Gen Bank accession number：KU131570） nucleocapsid protein gene for amplification of target sequence under isothermal condi-tions at 65℃ for 45 min. Amplification of RT-LAMP was monitored by the addition of calcein. A positive reaction was confirmed by changing from orange to yellow green under visual detection. The specificity of RT-LAMP assay was validated by cross-reaction with influenza virus A,influenza virus B and other human coronaviruses. Results The sensitivity was evaluated by serial dilution of HCoV-OC43 RNA from 5.6 ×10~6 to 5.6×10~0 copies per reaction. A set of primers with high specificity was finally settled through primer screening,which only reacted with HCoV-OC43 RNA and manifested with color changes. Compared with conventional Real-time RT-PCR method,The RT-LAMP assay could achieve 5.6 copies per reaction with a higher specificity in shorter time. Conclusion The colorimetric RT-LAMP assay could provide point-of-care detection of HCoV-OC43,and it has the potential to be applied at ports as well as at the primary medical institutions and epidemic areas.