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首页> 美国卫生研究院文献>Springer Open Choice >Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum
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Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum

机译：接收器工作特征曲线分析在实时PCR中确定LOQ：在镰刀菌和拟南芥的qPCR分析中的应用

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Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR.
机译：实时PCR（qPCR）是定量宿主组织中病原生物量的主要技术，但尚无通用方法来确定qPCR中的定量限（LOQ）和检测限（LOD）。为此，我们建议在接收器工作特性（ROC）曲线分析的背景下使用Youden指数。 LOQ被定义为最大化敏感性和特异性总和的靶DNA量。 LOD定义为以低于给定阈值的假阴性率扩增的最低目标DNA量。我们将此概念应用于玉米粒中镰孢镰刀菌和镰刀菌DNA的qPCR分析。以PCR产物的熔解曲线分析为特征的尖峰基质和野外样品用作真阳性和真阴性的来源。在分析的敏感性和特异性分析的基础上，我们估计网状拟南芥的LOQ值分别为加标基质的DNA的0.11pg DNA和现场样品的DNA的0.62pg DNA。掺入基质的F. proliferatum的LOQ值为0.03pg，现场样品的LOQ值为0.24pg。平均LOQ值对应于网状拟南芥的大约8个基因组和增殖拟南芥的3个基因组。我们证明了针对定性诊断开发的ROC分析概念可用于确定定量PCR的性能参数。

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