Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics, F76000, Normandy Center for Genomic and Personalized Medicine, Rouen, France.
Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics and CNR-MAJ, F 76000, Normandy Center for Genomic and Personalized Medicine, Rouen, France.
Clin Chem. 2019 Sep;65(9):1153-1160. doi: 10.1373/clinchem.2019.304246. Epub 2019 Jul 10.
Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes.
We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis.
UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods.
The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.
罕见的拷贝数变异（CNVs）是遗传疾病的主要原因。需要简单的靶向方法来确认和分离分析。我们开发了一种基于数字 PCR（dPCR）和通用锁核酸（LNA）水解探针的简单通用 CNV 检测方法。
我们分析了罗氏通用探针库（UPL）中的 90 个 LNA 水解探针的映射。对于每个 CNV，最佳引物和 LNA 探针的选择几乎是自动化的；探针可在多个检测中重复使用，每个 dPCR 检测都包括 CNV 扩增子和参考扩增子。我们在 93 个小和大的 CNVs 上评估了该检测方法的性能，并进行了成本效益分析。
UPL-LNA 探针在人类基因组上出现了近 20000000 次，均匀分布，平均间隔为 156bp。该检测方法准确地检测到了所有 93 个 CNVs，除了一个（<200bp），变异系数<10%。与其他所有方法相比，该检测方法的成本效益更高。
通用 dPCR CNV 检测方法简单、稳健且具有成本效益，因为它结合了通用探针和终点 PCR 允许的简单设计、同一反应中目标与参考的相对定量的优势，以及 LNA 水解探针的高灵活性。这种方法应该是基因组医学的有用工具，它需要简单的方法来解释和分离分析基因组变异。
