Institute of Basic Medical Sciences, Meizhou People's Hospital, Meizhou Academy of Medical Sciences, Meizhou, 514031, People's Republic of China.
Guangdong Engineering Technological Research Center of Clinical Molecular Diagnosis and Antibody Drugs, Meizhou Academy of Medical Sciences, Meizhou, 514031, People's Republic of China.
Drug Des Devel Ther. 2024 Nov 3;18:4889-4899. doi: 10.2147/DDDT.S491972. eCollection 2024.
The polymorphism of the solute carrier organic anion transporter family member gene exerts a marked influence on drug transport, thus playing a pivotal role in personalized drug dosing. This study endeavours to establish a rapid, precise, and straightforward method for detecting genetic variants utilizing Duplex Fluorescence Melting Curve Analysis (DFMCA).
Whole blood samples were collected from 54 individuals from Meizhou People's Hospital (2023.01-2023.03), with a mean age of 58.90 years (SD = 7.86), including 28 men and 26 women. DNA was extracted from these samples and subjected to PCR amplification targeting two allelic regions. Primers, fluorescent probes, and corresponding allelic target sequences were designed specifically for two common polymorphisms (rs2306283 and rs4149056). The functionality of the fluorescent probes in binding to their respective allelic targets was verified using melting curve analysis, enabling the identification of distinct melting temperatures for different genotypes. Subsequently, DFMCA was employed to differentiate genotypes based on the melting temperature shifts of the corresponding fluorescent probes. The sensitivity, accuracy, and consistency of the method were evaluated, with sequencing validation performed on a subset of samples.
DFMCA facilitated the concurrent detection and accurate genotyping of both polymorphisms within 2 hours, demonstrating concordance with sequencing results from randomly selected samples. Importantly, stable detection performance was achieved for human genomic DNA at concentrations ≥ 3.125 ng. In a cohort comprising Han Chinese individuals from southern China, the allele frequencies for rs2306283 (A: 28.7%, G: 71.3%) and rs4149056 (T: 88.89%, C: 11.11%) concurred well with previous studies in the Han Chinese population.
The SNP typing system utilizing DFMCA technology presents advantages in terms of speed, ease of use, accuracy, and cost-effectiveness, making it a suitable tool.
溶质载体有机阴离子转运家族成员基因的多态性对药物转运有显著影响，因此在个体化药物剂量中起着关键作用。本研究旨在建立一种快速、精确、直接的利用双荧光熔融曲线分析（DFMCA）检测基因变异的方法。
收集 54 例来自梅州市人民医院的个体（2023.01-2023.03）的全血样本，平均年龄 58.90 岁（SD=7.86），包括 28 名男性和 26 名女性。从这些样本中提取 DNA，并进行针对两个等位基因区域的 PCR 扩增。针对两个常见的多态性（rs2306283 和 rs4149056）设计了引物、荧光探针和相应的等位基因靶序列。使用熔融曲线分析验证了荧光探针与各自等位基因靶序列结合的功能，从而确定了不同基因型的不同熔融温度。随后，根据相应荧光探针的熔融温度变化，采用 DFMCA 区分基因型。评估了该方法的灵敏度、准确性和一致性，并对一部分样本进行了测序验证。
DFMCA 在 2 小时内实现了两种多态性的同时检测和准确基因分型，与随机选择样本的测序结果一致。重要的是，在浓度≥3.125ng 的人基因组 DNA 中实现了稳定的检测性能。在中国南方汉族人群的队列中，rs2306283（A：28.7%，G：71.3%）和 rs4149056（T：88.89%，C：11.11%）的等位基因频率与之前汉族人群的研究结果一致。
利用 DFMCA 技术的 SNP 分型系统在速度、易用性、准确性和成本效益方面具有优势，是一种合适的工具。
