High-throughput blood sample preparation for single nucleotide polymorphism genotyping in less than 25 min
High-throughput blood sample preparation for single nucleotide polymorphism genotyping in less than 25 min
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在 25 分钟内完成用于单核苷酸多态性基因分型的高通量血样制备
DOI：
10.1016/j.talanta.2018.08.051
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发表时间：
2018
期刊：
影响因子：
6.1
通讯作者：
Chen Wenwen
中科院分区：
文献类型：
--
作者：
Wang Jidong;Zheng Jiaying;Zhang Shaohua;Du Jihui;Chen Yongxin;Liu Xiaolei;Zhang Huisheng;Jiang Xingyu;Chen Wenwen
临床上迫切需要简单、快速、高通量的单核苷酸多态(SNP)基因分型前处理方法。然而，现有的SNP基因分型方法都需要DNA纯化步骤，劳动强度大，耗时长。我们开发了一种结合全血裂解和定量聚合酶链式反应的SNP基因分型方法，并在140名献血者的血液样本中验证了我们的方法的实用性，其中包括40名健康献血者的血液样本，以及100名白细胞计数低、血清尿酸或甘油三酯水平高的献血者的样本。与SNP基因分型的金标准Sanger测序相比，我们的方法在敏感性和特异性方面表现出100%的一致性。此外，我们的方法可以在25 分钟内从48个样本中获得可扩增的DNA(据我们所知这是最快的)。血样即使白细胞数低，血尿酸或甘油三酯水平高，也不影响本方法的敏感性和特异性。我们的研究表明，WBL和qPCR基因分型相结合可以作为一种高通量和稳健的常规临床SNP基因分型方法。
Straightforward, rapid and high-throughput pretreatment for single nucleotide polymorphisms (SNP) genotyping is critically needed in clinical practice. However, all existing SNP genotyping methods require DNA purification step, which is labor-intensive and time-consuming. We develop a protocol for SNP genotyping by combining whole blood lysis (WBL) with qPCR and justify the practicality of our method in blood samples from 140 donors, including 40 samples from healthy donors, and 100 samples from donors with either low white blood cell counts, high level of serum uric acid or triglyceride. When compared with Sanger sequencing, the gold standard for SNP genotyping, our method exhibits a 100% consistency in the aspect of sensitivity and specificity. In addition, our method can obtain amplifiable DNA within 25 mins (which is the fastest to the best of our knowledge) from 48 samples. The blood samples, even with low white blood cell counts, high level of serum uric acid or triglyceride could not affect the sensitivity and specificity of our method. Our study demonstrates that the combination of WBL and qPCR genotyping can serve as a high-throughput and robust approach for routine clinical SNP genotyping.