Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish
Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish
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DOI：
10.1186/1746-6148-9-183
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发表时间：
2013-09-16
影响因子：
2.6
通讯作者：
Marshall, Sergio H.
中科院分区：
文献类型：
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作者：
Sepulveda, Dagoberto;Bohle, Harry;Marshall, Sergio H.
背景：早期病原检测是水产养殖疾病控制的关键。因此，准确的诊断程序是必须的。多年来，由于其速度、特异性、灵敏度、可重复性和通量，实时PCR一直是诊断的支柱；因此，实时PCR是一个有待改进的目标。然而，为了验证一种新的诊断工具，必须正确设置检测方法，包括适当的内源性控制来评估样品的数量和质量，并检测可能的样品降解。本工作旨在设计一种独特的RT-qPCR检测方法，用于智利饲养的三种鲑鱼的病原体检测。该方法使用伸长因子1作为单一内源对照，从而避免了需要多个内源对照，以及多个验证和不可比较的质量控制参数。结果：Salmo salar， Oncorhynchus mykiss和Oncorhynchus kisutch样品的体内和体外分析表明，当准确选择引物时，可以设计出一种新的RT-qPCR方法，在三个物种之间具有相似且可重复的Ct值。当最初为Salmo salar设计的测定方法在Oncorhynchus属的两个物种的样本中进行测试时，情况正好相反。结论：在这里，我们报告了一种准确的跨物种RT-qPCR检测的设计和评估，该检测使用伸长因子1 α (ELF1 α)基因作为内源性对照，适用于从智利饲养的三种鲑鱼中获得的样本的诊断目的。
Background: The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters.Results: The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1 alpha) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus.Conclusions: Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1 alpha) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile.