特异性引物 PCR 片段上的铜纳米团簇具有磁性捕获，用于 BCR-ABL1 基因中 T315I 单核苷酸变异的无标记荧光检测,Analyst

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特异性引物 PCR 片段上的铜纳米团簇具有磁性捕获，用于 BCR-ABL1 基因中 T315I 单核苷酸变异的无标记荧光检测
Analyst ( IF 3.6 ) Pub Date : 2022-11-17 , DOI: 10.1039/d2an01433e
Ke-Peng Lai, Yu-Chen Su, Bo-Siang Fu, Kung-Hung Lin, Hwang-Shang Kou, Chun-Chi Wang

在这项研究中，设计了一个简单易行的程序，使用在特定引物 PCR 片段上全部或不形成双链 DNA 模板铜纳米团簇，以荧光方式鉴定BCR – ABL1基因上的 T315I 单核苷酸变体。慢性粒细胞白血病 (CML)，一种由BCR – ABL1引起的疾病酪氨酸激酶的融合，因 T315I 突变而众所周知，该突变导致酪氨酸激酶抑制剂 (TKI) 由于药物结合位点的替代结构而受到抵抗。因此，它是临床检测的重要单核苷酸变异。本研究仅设计了特异性功能引物，并用特异性限制性内切酶对来自T315I突变位点的野生基因型进行消化，然后使用磁珠捕获不同的消化产物。最终产品将允许通过荧光传感用于检测 T315I 突变的双链 DNA 模板铜纳米团簇的全部或全部形成。该研究已成功应用于鉴定野生和突变纯合子以及 T315I 突变的突变体/野生杂合子。预计该分析系统未来可作为T315I突变临床诊断的工具，应用于CML患者的真实样本。

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Copper nanoclusters on specific-primer PCR fragments with magnetic capture for the label-free fluorescent sensing of the T315I single nucleotide variant in the BCR–ABL1 gene

In this study, a simple and facile procedure using the all or none formation of double-stranded DNA-templated copper nanoclusters on specific-primer PCR fragments was designed to fluorescently identify the T315I single nucleotide variant on the BCR–ABL1 gene. Chronic myeloid leukaemia (CML), a disease caused by the BCR–ABL1 fusion of tyrosine kinase, is well known for the T315I mutation that causes tyrosine kinase inhibitors (TKIs) to be resisted due to the alternative structure of the drug-binding site. Therefore, it is an important single nucleotide variant for clinical detection. In this study, only specific functional primers and the digestion of the wild genotype from the T315I mutation site with specific restriction enzymes were designed, and the different digested products could then be captured using magnetic beads. The final products would allow for fluorescent sensing via the all or none formation of double-stranded DNA-templated copper nanoclusters for the detection of the T315I mutation. This study has been successfully applied for identifying wild and mutant homozygotes and the mutant/wild heterozygote of the T315I mutation. It is expected that this analytical system can serve as a tool for the clinical diagnosis of T315I mutations and be applied to real samples of CML patients in the future.

更新日期：2022-11-17

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