新型一体化数字 PCR 仪器上开发和验证用于脊髓性肌萎缩症（SMA）基因分型的 4 色多重荧光定量 PCR 检测方法。

Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument.

机构信息

Combinati Inc., 2450 Embarcadero Way, Palo Alto, CA, 94303, USA.

Center for Applied Clinical Genomics, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, USA.

出版信息

Sci Rep. 2020 Nov 16;10(1):19892. doi: 10.1038/s41598-020-76893-7.

Digital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

数字 PCR（dPCR）技术已被证明在检测拷贝数变异（CNV）方面具有高度的灵敏性和准确性。然而，目前尚未报道用于测量脊髓性肌萎缩症（SMA）样本中 SMN1 和 SMN2 拷贝数的高阶多重 dPCR 检测方法。本文介绍了一种在具有五个光学通道的完全集成的 dPCR 仪器上运行的快速多重 SMA dPCR 基因分型检测方法。该基于水解探针的多重 dPCR 检测方法定量检测 SMN1、SMN2 和总 SMN（SMN1+SMN2），同时将 RPPH1 基因用作内部参考对照。该四重检测方法用经过特征分析的对照 DNA 样本进行了评估，并使用先前发表的研究中的 15 个盲临床样本进行了验证。SMN1 和 SMN2 拷贝数与对照和盲样的先前结果完全一致。基于 dPCR 的 SMA 拷贝数测定在 90 分钟内完成，其具有与实时定量 PCR（qPCR）相同的免提工作流程。总之，本文提出了一种基于新型数字 PCR 平台的简单高阶多重解决方案，以满足 SMA 基因分型和预后日益增长的需求。