Background Phytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species. Quantitative real-time PCR (qPCR) is one of the most commonly used techniques for pathogen detection, especially for pathogens that cannot be cultivated outside their host like phytoplasma. PCR analysis requires the purification of total DNA from the sample and subsequent amplification of pathogen DNA with specific primers. The purified DNA contains mainly host DNA and only a marginal proportion is of phytoplasmal origin. Therefore, detection of phytoplasma DNA in a host DNA background must be sensitive, specific and reliable and is highly dependent on the quality and concentration of the purified DNA. DNA quality and concentration and the presence of PCR-inhibitors therefore have a direct impact on pathogen detection. Thus, it is indispensable for PCR-based diagnostic tests to validate the DNA preparation and DNA integrity before interpreting diagnostic results, especially in case that no pathogen DNA is detected. The use of an internal control allows to evaluate DNA integrity and the detection of PCR-inhibiting substances. Internal controls are generally host-specific or limited to a defined group of related species. A control suitable for the broad range of phytoplasma hosts comprising different insect and plant species is still missing. Results We developed a primer and probe combination that allows amplification of a conserved stretch of the eukaryotic 28S rDNA gene. The developed endogenous qPCR control serves as a DNA quality control and allows the analysis of different eukaryotic host species, including plants, insects, fish, fungi, mammals and human with a single primer/probe set in single- or multiplex assays. Conclusions Quality and performance control is indispensable for pathogen detection by qPCR. Several plant pathogens are transmitted by insects and have a broad range of host species. The newly developed endogenous control can be used with all so far tested eukaryotic species and since multiplexing is possible, the described primer and probe set can be easily combined with other PCR-based pathogen detection systems.

中文翻译：

---

开发真核 DNA 样品的通用内源 qPCR 对照。

背景 植原体是专性细胞内植物病原细菌，可感染多种植物物种并通过不同昆虫物种传播。实时定量 PCR (qPCR) 是病原体检测最常用的技术之一，特别是对于植原体等无法在宿主体外培养的病原体。PCR 分析需要从样品中纯化总 DNA，然后使用特定引物扩增病原体 DNA。纯化的 DNA 主要含有宿主 DNA，只有极少数是植质来源的。因此，宿主 DNA 背景中植原体 DNA 的检测必须灵敏、特异且可靠，并且高度依赖于纯化 DNA 的质量和浓度。因此，DNA 质量和浓度以及 PCR 抑制剂的存在对病原体检测有直接影响。因此，基于 PCR 的诊断测试在解释诊断结果之前验证 DNA 制备和 DNA 完整性是必不可少的，特别是在未检测到病原体 DNA 的情况下。使用内部对照可以评估 DNA 完整性并检测 PCR 抑制物质。内部控制通常针对宿主特定或仅限于一组确定的相关物种。仍然缺乏适合包括不同昆虫和植物物种的广泛植原体宿主的控制。结果我们开发了一种引物和探针组合，可以扩增真核 28S rDNA 基因的保守片段。开发的内源 qPCR 控制可作为 DNA 质量控制，并允许在单次或多重测定中使用单一引物/探针组分析不同的真核宿主物种，包括植物、昆虫、鱼类、真菌、哺乳动物和人类。结论 质量和性能控制对于 qPCR 病原体检测是必不可少的。几种植物病原体通过昆虫传播，并且具有广泛的宿主物种。新开发的内源对照可用于迄今为止测试的所有真核物种，并且由于可以进行多重检测，因此所描述的引物和探针组可以轻松地与其他基于 PCR 的病原体检测系统相结合。