BACKGROUND Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA. METHODS We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106-1); (ii) diluted template DNA from pure oocysts (×10-×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105-1 and 5 × 104-50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10-×1000). RESULTS Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations. CONCLUSIONS For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

中文翻译：

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评估细胞培养的小隐孢子虫卵囊和自由悬浮的卵囊 DNA 含量之间的差异及其作为 qPCR 中 DNA 标准品的适用性。

背景技术虽然有更现代的方法可用，但定量 PCR (qPCR) 具有可重复性、灵敏性和特异性，并且许多实验室都可以轻松获得仪器和专业知识。因此，qPCR 在隐孢子虫研究中的应用已经很成熟，并且仍然被全球研究人员广泛使用。该方法取决于不同浓度标准品的生成，以生成随后用于 DNA 定量的标准曲线。方法 我们评估了四种类型的 DNA 模板，用于在涉及隐孢子虫属的药物筛选研究中生成标准曲线： (i) 连续稀释的隐孢子虫卵囊 (106-1)；(ii) 来自纯卵囊的稀释模板DNA（106个卵囊DNA模板的×10-×106稀释）；(iii) 在人回盲部腺癌 (HCT-8) 细胞（105-1 和 5 × 104-50）中孵育的卵囊；(iv) 来自细胞培养物孵化的寄生虫 (×10-×1000) 的稀释 DNA 模板 (5 × 104)。结果 细胞培养物和纯卵囊悬浮液 DNA 模板的连续稀释比细胞培养物衍生的标准品产生更好的线性，106 个卵囊的稀释液表现出与 106 个卵囊 DNA 模板稀释液相似的定量周期 (Cq) 值。相比之下，细胞培养物孵育的卵囊表现出比同等的自由悬浮卵囊和来自细胞培养物衍生的和自由悬浮卵囊的多种浓度的稀释DNA模板显着更高的DNA含量。结论 对于许多涉及隐孢子虫的研究，只需要相对 DNA 含量，因此，自由悬浮的卵囊和稀释的 DNA 模板（来自细胞培养衍生的标准品或自由悬浮的卵囊）提供的卓越线性将允许在每个样本中进行更准确的相对定量。化验。细胞培养标准中的寄生虫分裂可能解释了所发现的较高 DNA 含量。因此，这些标准有可能更准确地反映细胞培养测定中的 DNA 含量，尽管有更现代的方法可用于绝对定量，即微滴数字 PCR (ddPCR)，但在可预见的未来，qPCR 的普遍存在将鼓励进一步研究在这些标准中观察到的线性度降低，例如不同的卵囊接种密度、非线性生长速率和测定效率。