School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100, Shi-chuan 1st Rd, Kaohsiung, 807, Taiwan, Republic of China.
Department of Surgery, Division of General Surgery, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, Republic of China.
Mikrochim Acta. 2023 Aug 31;190(9):375. doi: 10.1007/s00604-023-05950-5.
A simple, rapid, and highly efficient fluorescent detection technique without PCR through dual-probe ligation with the genetic capture of magnetic beads and reported probe was developed for determination of epidermal growth factor receptor (EGFR) gene exon 19 deletions. The EGFR exon 19 deletion mutation makes up 48% of all mutations associated with anti-tyrosine kinase inhibition sensitivity, and thus, the EGFR nucleotide variant is very important in clinical diagnosis. In this approach, the dual-probe ligation was designed to target exon 19 deletion. The magnetic genetic captured system was then applied to capture the successful dual-probe ligation. Thereafter, a reporter probe which is coupled with 6-fluorescein amidite (6-FAM) was introduced to hybridize with dual-probe ligation product on the surface of streptavidin magnetic beads, and finally, the supernatant was taken for fluorescence measurements for distinguishing mutant types from wild types. After optimization (the RSD of the fluorescent intensity was less than 4.5% (n = 3) under the optimal condition), 20 blind DNA samples from the population were analyzed by this technique and further confirmed by direct sequencing. The results of our assay matched to those from direct sequencing data, evidencing that the developed method is accurate and successful. These 20 blind DNA samples were diagnosed as wild and then spiked with different percentages of the mutant gene to quantify the ratio of the wild and mutant genes. This strategy was also successfully applied to quantify the ratio of the wild and mutant genes with good linearity at the λ/λ of 480 nm/520 nm (r = 0.996), and the limit of detection reached 1.0% mutant type. This simple fluorescent detection of nucleotide variants shows its potential to be considered a tool in biological and clinical diagnosis. Importantly, this strategy offers a universal detection capability for any kind of mutation (point, deletion, insertion, or substitution) in a gene of interest.
一种简单、快速、高效的荧光检测技术，无需 PCR，通过双探针连接与磁性珠的基因捕获和报告探针相结合，用于测定表皮生长因子受体（EGFR）基因外显子 19 缺失。EGFR 外显子 19 缺失突变构成了与抗酪氨酸激酶抑制敏感性相关的所有突变的 48%，因此，EGFR 核苷酸变体在临床诊断中非常重要。在这种方法中，双探针连接设计用于靶向外显子 19 缺失。然后应用磁性基因捕获系统捕获成功的双探针连接。此后，引入与 6-荧光素胺（6-FAM）偶联的报告探针与链霉亲和素磁性珠表面上的双探针连接产物杂交，最后，取上清液进行荧光测量，以区分突变型与野生型。经过优化（在最佳条件下，荧光强度的 RSD 小于 4.5%（n = 3）），通过该技术分析了 20 个来自人群的盲 DNA 样本，并通过直接测序进一步确认。我们的检测结果与直接测序数据一致，证明了该方法的准确性和成功性。这 20 个盲 DNA 样本被诊断为野生型，然后用不同百分比的突变基因进行了掺入，以定量野生型和突变型基因的比例。该策略还成功地应用于以 480nm/520nm 的 λ/λ 值（r = 0.996）进行定量分析，检测限达到 1.0%突变型。这种简单的核苷酸变体荧光检测显示出其在生物和临床诊断中被认为是一种工具的潜力。重要的是，该策略为感兴趣基因中的任何类型的突变（点突变、缺失、插入或取代）提供了通用的检测能力。
