Abstract：
Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.
摘要：
双生病毒科和伊夫病毒科中的病毒是对西方蜜蜂（Apismellifera）和本地蜜蜂物种的主要威胁。聚合酶链反应（PCR）是蜜蜂病原体检测的金标准。然而,在单重PCR反应中高通量筛选蜜蜂病毒感染是麻烦的,并且受到所需的大量样品RNA的限制。因此，有必要开发一种灵敏、特异的多重PCR检测方法同时筛选多种病毒。这里,我们报道了一步多重逆转录定量聚合酶链反应（RT-qPCR）检测传粉者物种中常见的四种病毒的发展。本研究中描述的优化的多重RT-qPCR方案允许同时检测两种双螺旋病毒（以色列急性麻痹病毒和黑皇后细胞病毒）和两种iflavirus（Sacbrood病毒和Deformedwing病毒），具有与单重检测相当的高效率和特异性。该测定提供了广泛的检测和定量，并且本研究中的病毒定量结果与使用单重检测测定法的其他研究中的结果相似。该方法对于从产生少量RNA的小体态昆虫物种产生数据将特别有用。
