Wildlife Epidemiology Laboratory, College of Veterinary Medicine at University of Illinois, Urbana, IL 61802, USA.
Department of Comparative, Diagnostic, and Population Medicine, University of Florida, Gainesville, FL 32611, USA.
Cytokine. 2019 Jul;119:16-23. doi: 10.1016/j.cyto.2019.02.011. Epub 2019 Mar 8.
In response to viral pathogens, a host releases pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) and anti-inflammatory cytokines such as interleukin-10 (IL-10). While several approaches exist to measure cytokine responses, evaluating gene transcription through reverse transcription quantitative polymerase chain reaction (RT-qPCR) provides a fast, reproducible, and sensitive method for quantifying this response. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the quantification of red-eared slider (Trachemys scripta elegans) and eastern box turtle (Terrapene carolina carolina) cytokines: IL-1β, TNFα, IL-10 and the reference gene β-actin. RNA was isolated from the buffy coat layer of whole blood, comprised mainly of circulating leukocytes, and complimentary DNA (cDNA) was produced. Conventional PCR was performed to obtain cytokine mRNA sequences, products were sequenced, and a hydrolysis probe-based RT-qPCR assay was designed for each cytokine. Standard curves were generated using the target gene sequences cloned within a plasmid. Efficiencies for each assay were between of 85-110%, R > 0.98, and limits of detection of 10-100 copies per reaction. The initial samples used to identify the novel target sequences were then used to evaluate the performance of the qPCR assays. Consistent transcription of beta actin across individuals in both species and measurable transcription of IL-1β, TNF-α, and IL-10 transcript targets in individuals of both species were observed. The assays are a novel technique in chelonians to evaluate host innate immune response.
针对病毒病原体，宿主会释放促炎细胞因子，如白细胞介素-1β（IL-1β）和肿瘤坏死因子α（TNF-α），以及抗炎细胞因子，如白细胞介素-10（IL-10）。虽然有几种方法可以测量细胞因子反应，但通过反转录定量聚合酶链反应（RT-qPCR）评估基因转录为定量测量这种反应提供了一种快速、可重复和敏感的方法。本研究的目的是开发一种有效和敏感的 RT-qPCR 检测方法，用于定量测定红耳滑龟（Trachemys scripta elegans）和东部箱龟（Terrapene carolina carolina）的细胞因子：IL-1β、TNFα、IL-10 和参考基因β-肌动蛋白。从主要由循环白细胞组成的全血的白细胞层中分离 RNA，并生成互补 DNA（cDNA）。进行常规 PCR 以获得细胞因子 mRNA 序列，对产物进行测序，并为每个细胞因子设计基于水解探针的 RT-qPCR 检测方法。使用克隆在质粒内的靶基因序列生成标准曲线。每个检测的效率在 85-110%之间，R>0.98，检测限为每个反应 10-100 个拷贝。然后，使用最初用于鉴定新靶序列的样本来评估 qPCR 检测方法的性能。在两个物种的个体中，β肌动蛋白在个体间的转录具有一致性，并且可以在两个物种的个体中检测到 IL-1β、TNF-α 和 IL-10 转录靶标的可测量转录。该检测方法是评估龟类宿主固有免疫反应的新技术。
