School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1.
J Invertebr Pathol. 2010 Oct;105(2):151-5. doi: 10.1016/j.jip.2010.06.001. Epub 2010 Jun 4.
Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R(2)=0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.
正确鉴定微孢子虫、蜂微孢子虫（Nosema apis）和蜜蜂微孢子虫（Nosema ceranae）是研究和控制蜜蜂微孢子虫病（Apis mellifera）的关键。本研究比较了一种快速 DNA 提取方法与多重 PCR 联合使用种特异性引物扩增 16S rRNA 基因与先前发表的需要孢子萌发缓冲液和 DNA 提取试剂盒的检测方法。当使用孢子萌发-提取试剂盒方法时，需要 10 只或更多的蜜蜂来检测病原体，而新的提取方法可以在单个蜜蜂中检测到病原体。与孢子萌发-提取试剂盒方法相比，新方法对 N. ceranae 的检测灵敏度提高了约 4-8 倍。此外，与试剂盒相比，新方法处理样品所需的时间和成本更低。使用新的 DNA 提取方法，开发了一种使用三重 PCR 检测孢子的方法，该方法共扩增蜜蜂的核糖体蛋白基因 RpS5 和 N. apis 和 N. ceranae 的 16S rRNA 基因。通过将相对条带强度与 23 个样本的显微镜下每个蜜蜂的孢子数进行比较，确定了这种半定量 PCR 的准确性，观察到高度相关性（R(2)=0.95）。这种 Nosema 孢子定量方法表明，PCR 可以检测低至 100 个孢子/只的孢子。与以前发表的基于 PCR 的标准检测方法相比，新的半定量三重 PCR 检测方法更敏感、经济、快速、简单、可靠，对于没有实时 PCR 的实验室将非常有用。
