黄热病毒可视化RT-LAMP检测方法的建立

Development of a visual RT-LAMP method for yellow fever virus detection

目的建立一种根据颜色变化判断结果的黄热病毒可视化RT-LAMP快速核酸检测方法。方法针对112株目前GeneBank上所有基因亚型黄热病毒株的5’端非编码区保守性核苷酸序列,设计黄热病毒特异性LAMP引物,建立黄热病毒可视化RT-LAMP检测方法。评价该方法的特异性、灵敏性,并用临床样本和模拟样本进行应用性验证。结果建立的RT-LAMP方法有较高的特异性,与登革病毒、裂谷热病毒、西尼罗病毒、乙脑病毒及基孔肯雅病毒无交叉反应;用体外转录的RNA模板评估,其灵敏性可达102拷贝/μl;用口岸发热病例血清样本验证,该方法可检出血清中的黄热病毒。结论建立的黄热病毒可视化RT-LAMP检测方法操作简便、快速、特异性好,可应用于黄热病毒的现场检测。

Objective To develop a visual RT-LAMP rapid nucleic acid detection method for yellow fever virus based on color changes judgement.Methods A yellow fever virus-specific LAMP primer was designed for the conserved nucleotide sequence of the 5’non-coding region of all the gene subtypes of the 112 strains in GeneBank,and yellow fever virus visual RT-LAMP detection method was established.The specificity and sensitivity of the method were evaluated and clinical validation was performed by using clinical samples and simulated clinically in-fected blood samples.Results The established RT-LAMP detection method had high specificity and without crossreaction with dengue virus,Rift Valley fever virus,West nile virus,Japanese encephalitis virus and chikungunya virus.The sensitivity of the positive RNA template in vitro was up to 102 copies/μl.It was tested by serum samples collected from the fever cases at ports and the result showed that the method could detect the yellow fever virus in the serum.Conclusion The visual RT-LAMP method for yellow fever virus detection established in this study is simple,rapid and specific,and can be applied to the on-site detection of yellow fever virus.