Swiss Bee Research Centre, Agroscope Liebefeld-Posieux Research Station ALP, 3003 Bern, Switzerland.
J Virol Methods. 2011 Jun;174(1-2):150-2. doi: 10.1016/j.jviromet.2011.03.029. Epub 2011 Apr 5.
Molecular diagnoses of pathogens via ribonucleic acid (RNA) signatures are used widely in honey bee pathology. Such diagnoses can be compromised by ubiquitous and endogenous RNA-degrading enzymes activated after the death of sampled bees. RNA degradation can be minimized by storage at ultra-cold temperatures or by immersion in high-salt buffers. However, these methods are not always available in the field or are costly, driving a search for alternative methods to store and transport bees for RNA analyses. While the impact of storage conditions on RNA integrity has been evaluated, the tolerance of standard RT-qPCR diagnostic methods of honey bee pathogens for suboptimal collection and storage is unknown. Given the short regions of RNA used for pathogen diagnosis (generally amplified regions of 100-200 nucleotides), it is conceivable that even degraded RNA will provide a template for precise diagnosis. In this study, the impact of the two most convenient sample storage and handling methods (+4°C and ambient temperature) for identifying honey bee virus infections was evaluated by RT-qPCR. The aim was to streamline the methods needed to collect, transport, and store honey bee samples destined for pathogen diagnosis. The data show that samples held at room temperature for times anticipated for sample transport for up to 5 days are suitable for diagnosis of two of the most common and prevalent honey bee viruses, deformed wing virus (DWV) and black queen cell virus (BQCV). The results will be useful for the standardisation of sampling methods across countries and laboratories.
通过核糖核酸 (RNA) 特征对病原体进行分子诊断在蜜蜂病理学中被广泛应用。然而，这些诊断可能会受到无处不在的和内源性的 RNA 降解酶的影响，这些酶在采样蜜蜂死亡后被激活。通过在超低温下储存或在高盐缓冲液中浸泡可以最大程度地减少 RNA 降解。然而，这些方法在现场并不总是可用的，或者成本很高，因此需要寻找替代方法来储存和运输蜜蜂以进行 RNA 分析。虽然已经评估了储存条件对 RNA 完整性的影响，但标准 RT-qPCR 诊断方法对蜜蜂病原体的不理想收集和储存的容忍度尚不清楚。鉴于用于病原体诊断的 RNA 区域较短（通常为 100-200 个核苷酸的扩增区域），可以想象，即使是降解的 RNA 也将为精确诊断提供模板。在这项研究中，通过 RT-qPCR 评估了两种最方便的样本储存和处理方法（+4°C 和环境温度）对鉴定蜜蜂病毒感染的影响。目的是简化收集、运输和储存用于病原体诊断的蜜蜂样本所需的方法。数据表明，在预计用于样本运输的时间内，在室温下保存长达 5 天的样本适合诊断两种最常见和流行的蜜蜂病毒，即变形翅膀病毒 (DWV) 和黑皇后细胞病毒 (BQCV)。研究结果将有助于在各国和实验室之间标准化采样方法。
