【摘要】 本研究利用3种方法对新城疫病毒(Newcastle disease virus,NDV)进行检测,并对这3种检测方法的灵敏度和特异性作出比较。根据新城疫病毒的融合蛋白基因(F基因)设计合成6条特异性引物,利用水浴LAMP法、PCR及LAMP实时浊度仪进行检测。通过对恒温水浴中的反应温度、反应时间及反应液中Mg2+浓度进行优化获得最佳反应条件,结果用凝胶电泳分析;用优化的温度进行LAMP实时浊度仪检测,同时都与PCR方法进行比较。结果显示,获得了最佳反应条件,水浴LAMP方法最低检测限是1.58pg,比PCR高100倍,而LAMP实时浊度仪检测灵敏度比水浴LAMP方法高10倍,最低检测限是0.158pg。3种方法对其他非新城疫病毒均无检出。结果表明,新城疫病毒水浴LAMP检测方法速度快、不需要高精密的仪器,而且具有灵敏度高、特异性强、操作简单等特点,有望在核酸扩增领域取代PCR技术,LAMP浊度仪法检测灵敏度更高,但是所需仪器和试剂比较昂贵。更多还原

【Abstract】 Newcastle disease viruses(NDV) were detected by three detecting tests and make a comparison of sensitivity and specificity about these tests.A total of six primers were designed and synthesized based on the fusion gene of NDV and were used for isothermal amplification of nucleic acid.The LAMP reaction conditions were optimized and nucleic acid amplificationwere detected in optimized temperature by LAMP turbidimetry.Detection of gene amplification could be accomplished by agarose gel electrophoresis.It was found that the detection limit of the water bath LAMP assay was 1.58 pg,it demonstrated 100-fold higher sensitivity than PCR,the detection limit of the LAMP turbidimetry assay was 15.8 ng and 10-fold higher sensitivity than the water bath LAMP,which had no cross-reaction with other viruses.The water bath LAMP was very simple and amplification can be obtained without extremely sophisticated instrumental.The water bath LAMP assay was extremely convenience,highly sensitive and specific and had potential replaced PCR in the field of nucleic acid amplification.LAMP turbidity assay was more sensitive,but required more expensive equipments and reagents.更多还原