甲型H1N1流感病毒快速诊断核酸试纸条的研制及应用

Development and application of a rapid nucleic acid diagnostic strip for influenza A(H1N1) virus

目的建立一种快速、灵敏度高、特异性强的核酸试纸条检测甲型H1N1流感病毒。方法根据2009年公布新型甲型H1N1流感病毒基因组序列设计合理的引物建立环介导恒温扩增体系,并在体系中加入含生物素的标记探针;在恒温下扩增45 min后产物与醋酸纤维棉片结合进行胶体金免疫试纸条显色反应,利用该方法检测76例确诊甲型H1N1流感病毒感染患者的咽部分泌物,同时采用卫生部公布的合格试剂盒进行对比试验。结果 76例确诊患者咽部分泌物经核酸试纸条检测全阳性,经对比试剂盒检测103拷贝/ml的标本进行10倍稀释后采用核酸试纸条检测结果仍能测出。结论核酸试纸条法检测甲型H1N1流感病毒具有快速、灵敏度高、特异性强的特点更适合于临床快速诊断。

OBJECTIVE To develop a rapid, sensitive and specific method for the detection of influenza A （H1N1） virus by the nucleic acid diagnostic strip. METHODS According to new type of genomic sequence of influenza A （H1N1） virus issued in 2009, a primers was constructed to create a system of loop mediated isothermal amplification, and a biotin labelled probe was spiked into this system. After amplified for 45 min, the resulting products were combined with cellulose acetate fiber for the color reaction on the immuno-colloidal gold test strips. This method was applied for 76 patients diagnosed with influenza A （H1N1） virus by using their pharyngeal secretion, and the results were compared with kit method issued by the Ministry of Health of the P. R. C. RESULTS All 76 results tested by the nucleic acid diagnostic strip were positive, and the samples with 10a Copies/ ml been detected by kit method can still be detected by the nucleic acid diagnostic strip method when diluted 10 times. CONCLUSION The method of nucleic acid diagnostic strip for influenza A（H1N1） virus detection shows the characteristics of rapidness, higher sensitivity and specificity, and is more suitable for the quick clinical diagnosis.