Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Veterinary Public Health and Food Science, University of Veterinary Medicine, Veterinaerplatz 1, 1210, Vienna, Austria.
Institute of Milk Hygiene, Milk Technology and Food Science, Department of Veterinary Public Health and Food Science, University of Veterinary Medicine, Veterinaerplatz 1, 1210, Vienna, Austria.
Sci Rep. 2018 May 29;8(1):8275. doi: 10.1038/s41598-018-26116-x.
Progressively more qPCR assays have been developed in recent years in numerous fields of application. These assays are routinely validated using calibration curves, but essential validation per se such as Poisson analysis is frequently neglected. However, validation is crucial for determination of resolution and quantitative and qualitative limits. The new test method PCR-Stop analysis presented in this work investigates assay performance during initial qPCR cycles. PCRs with one to five pre-runs are performed while the subsequent main qPCR runs reflect pre-run replication rates. Ideally, DNA doubles according to pre-runs, there is no variation between replicates and qPCR starts immediately at the first cycle with its average efficiency. This study shows two exemplary qPCR assays, both with suitable calibration curves and efficiencies. We demonstrated thereby the benefits of PCR-Stop analysis revealing quantitative and qualitative resolution of both assays, the limits of one of those assays and thus avoiding misinterpretations in qPCR analysis. Furthermore, data displayed that a well performing assay starts indeed with its average efficiency.
近年来，在许多应用领域中，越来越多的 qPCR 检测方法得到了发展。这些检测方法通常使用校准曲线进行常规验证，但至关重要的验证本身，如泊松分析，经常被忽视。然而，验证对于确定分辨率以及定量和定性极限是至关重要的。本文介绍了一种新的测试方法 PCR-Stop 分析，该方法用于研究初始 qPCR 循环期间的检测性能。进行了一到五个预运行的 PCR，而随后的主要 qPCR 运行反映了预运行的复制率。理想情况下，DNA 根据预运行呈指数增长，复制之间没有差异，qPCR 从第一个循环开始，其平均效率为初始值。本研究展示了两个示例 qPCR 检测方法，这两个方法都具有合适的校准曲线和效率。因此，我们证明了 PCR-Stop 分析的好处，该分析揭示了两种检测方法的定量和定性分辨率、其中一种检测方法的极限，从而避免了 qPCR 分析中的误解。此外，数据显示，性能良好的检测方法确实从其平均效率开始。
