Center for Medical Genetics and Prenatal Diagnosis, People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, People's Republic of China.
Research and Development Department, Shenzhen Biorain Technology Co., Ltd, Shenzhen, People's Republic of China.
Hematology. 2024 Dec;29(1):2365596. doi: 10.1080/16078454.2024.2365596. Epub 2024 Jun 12.
This study aimed to establish a droplet digital polymerase chain reaction (ddPCR) assay for South-East Asian (SEA) deletion based on a fully integrated digital PCR system DropXpert S6.
A total of 151 whole blood samples, 10 chorionic villus samples, and 17 amniotic fluid samples were collected, including 106 SEA heterozygotes, 43 normal individuals, 10 Hb Bart's hydrops details, and 19 SEA deletions combined with other genotypes.Genotypes of these samples were determined by the Gap-PCR method. We perform a series of optimizations of the ddPCR system to ensure the performance of the entire ddPCR reaction, such as droplet stability, fluorescence clustering, sensitivity, and accuracy.
Our assay exhibited 99.4% (177/178) accuracy compared with the Gap-PCR method, and the minimum detection limit of DNA was 0.1 ng/μL.Both targets have reliable linearity, R= 0.9999 for the α-thalassemia SEA deletion allele and R= 1 for the wild-type allele. The coefficient of variation for α-thalassemia SEA deletion allele detection at 2 and 10 ng/μL concentrations was 5.42% and 1.91%, respectively. In contrast, the coefficient of variation for wild-type allele detection was 4.06% and 1.83%, demonstrating its high quantitative accuracy. In addition, the DropXpert S6 PCR system showed some advantages over other ddPCR instruments, such as reducing testing costs, simplifying and automating the workflow.
The DropXpert S6 PCR system provided a highly accurate diagnosis for α-thalassemia SEA deletion and can be used to detect α-thalassemia as an alternative method.
本研究旨在建立一种基于完全集成的数字 PCR 系统 DropXpert S6 的东南亚（SEA）缺失的液滴数字聚合酶链反应（ddPCR）检测方法。
共采集 151 份全血样本、10 份绒毛膜样本和 17 份羊水样本，包括 106 例 SEA 杂合子、43 例正常个体、10 例 Hb Bart's 水肿细节和 19 例 SEA 缺失与其他基因型的组合。这些样本的基因型通过 Gap-PCR 方法确定。我们对 ddPCR 系统进行了一系列优化，以确保整个 ddPCR 反应的性能，如液滴稳定性、荧光聚类、灵敏度和准确性。
与 Gap-PCR 方法相比，我们的检测方法显示出 99.4%（177/178）的准确性，DNA 的最小检测限为 0.1ng/μL。两个靶标均具有可靠的线性，α-地中海贫血 SEA 缺失等位基因的 R=0.9999，野生型等位基因的 R=1。在 2 和 10ng/μL 浓度下，α-地中海贫血 SEA 缺失等位基因检测的变异系数分别为 5.42%和 1.91%。相比之下，野生型等位基因检测的变异系数为 4.06%和 1.83%，表明其具有较高的定量准确性。此外，DropXpert S6 PCR 系统与其他 ddPCR 仪器相比具有一些优势，例如降低检测成本、简化和自动化工作流程。
DropXpert S6 PCR 系统为α-地中海贫血 SEA 缺失提供了高度准确的诊断，可作为替代方法用于检测α-地中海贫血。
