基于TaqMan探针的蜜蜂囊状幼虫病病毒荧光PCR检测方法的建立和应用

为建立快速有效的蜜蜂囊状幼虫病检疫方法, 依据TaqMan荧光标记探针技术原理, 针对蜜蜂囊状幼虫病病毒保守序列, 设计出一对特异性引物和一条探针, 建立了一种快速检测蜜蜂囊状幼虫病病毒的荧光PCR方法。该方法对蜜蜂囊状幼虫病的检测具有较好的特异性, 与蜜蜂急性麻痹病病毒、蜜蜂慢性麻痹病病毒、蜜蜂残翼病病毒和黑蜂王台病病毒之间均无交叉反应。检测灵敏度可达1.0×10²拷贝/μL阳性质粒, 可对低病毒含量的样品进行准确检测。重复性和稳定性试验结果显示, 变异系数为1.6%, 说明该方法具有较好的重复性和稳定性。应用该方法对蜜蜂及蜂制品进行检测, 结果显示所建立的荧光PCR检测方法4 h内即可报告检测结果, 该方法具有快速、灵敏、特异及重复性好等优点, 适用于蜜蜂及其制品中蜜蜂囊状幼虫病病毒的快速检疫。

A real-time TaqMan PCR assay to detect sacbrood virus in honeybee and honeybee products

To establish a rapid and effective quarantine method of the honeybee sacbrood virus disease, we developed a real-time RT-PCR assay for detection of bee sacbrood disease using TaqMan probes. A pair of specific primers and a probe used in this assay were designed based on a highly conserved region in sacbrood bee virus. The assay was shown to be sensitive, detecting less than 1.0×10² copies/μL, and specific for the detection of sacbrood bee virus. Cross-reaction with acute bee paralysis virus, deformed wing virus and black queen cell virus was not observed. The coefficient of variation in the stability experiments was 1.6%. A reliable diagnostic result can be obtained just within 4 h. The assay proved to be a rapid, sensitive, specific and repetitive method for rapid detection of sacbrood bee virus from honeybee and honeybee products in quarantine.