阿尼昂尼昂病毒实时荧光定量RT-PCR检测方法的建立

The establishment of a fluorescent quantitative RT-PCR assay for the detection of O’nyong-nyong virus

目的建立阿尼昂尼昂病毒实时荧光定量RT-PCR检测方法。方法针对阿尼昂尼昂病毒nsP1基因设计特异性引物和探针,以珠海国际旅行卫生保健中心之前构建的MS2病毒样颗粒为标准品,采用一步法建立实时荧光定量RT-PCR检测方法,并对该方法的检测下限、特异性、精密度、抗干扰能力、符合率进行评价。结果建立的阿尼昂尼昂病毒实时荧光定量RT-PCR检测方法线性良好(线性方程Y=-3.746X+46.174,R2=0.988),检测下限为103copies/mL,低于之前文献报道的检测下限;能特异性地扩增阿尼昂尼昂病毒基因片段,而对其他相关病原体,包括同属于甲病毒属的基孔肯雅病毒、引起类似症状的登革病毒和马雅罗病毒、共用同一媒介宿主的疟原虫等无非特异性扩增;精密度良好,批内精密度测试时Ct值为32.92~35.40,变异系数CV为2.10%;批间精密度测试时Ct值为31.89~35.49,变异系数CV为3.41%;在存在溶血、脂血等干扰因素时Ct值与正常样本的Ct值差值分别为0.37、0.71,表明其能有效对抗溶血、脂血等干扰因素的影响;盲样检测时能检出4例阳性和12例阴性,同预期结果完全一致,阴阳性符合率可达100%。结论本研究建立的检测方法,灵敏度高、特异性好、精密度高、抗干扰能力强,可为阿尼昂尼昂病毒检测提供可行的方法。

Objective To establish a fluorescent quantitative real-time reverse transcript polymerase chain reaction (RT-PCR) assay for the detection of O’nyong-nyong virus.Methods Firstly,specific primers and probe were designed to target the conserved ns P1 gene of O’nyong-nyong virus.Then a one-step RT-PCR assay was established with MS2 phage based O’nyong-nyong virus-like particles as standards.Finally,we evaluated the lower limit of detection,specificity,precision,antiinterference ability and coincidence rate of the newly established assay.ResultsThe established RT-PCR assay for the detection of O’nyong-nyong virus showed good linearity,with the equation Y=-3.746X+46.174,R2=0.988.The lower limit of detection (LOD) was 103copies/m L,which was lower than previously reported LOD.The assay could detect O’nyong-nyong virus specifically,while other pathogens,including Chikungunya virus (which also belonged to alphavirus),Dengue virus and Mayaro virus (which shared similar symptoms),Plasmodium (which transmitted through the same kind of vectors),could not be detected.The assay also showed high precision performance.During within batch testing,the Ct value was between 32.92 and35.40,with a CV of 2.10%,while during between batch testing,the Ct value was between 31.89 and 35.49,with a CV of 3.41%.Hemeferroheme and lipids influenced the Ct value of the assay by 0.37 and 0.71,indicating the high anti-hemeferroheme and anti-lipids ability.Moreover,the assay could detect exactly 4 positive samples and 12 negative samples from the blind samples,with a coincidence rate of 100%.Conclusion The established assay showed high sensitivity,good specificity,high precision performance and high anti-influence ability,which could provide a feasible method for the detection of O’nyong-nyong virus.