Rapid Detection of SLCO1B1 Polymorphisms Using Duplex Fluorescence Melting Curve Analysis: Implications for Personalized Drug Dosing in Clinical Settings

Zhikang Yu,1,2,* Zifang Shang,1,2,* Qingyan Huang,3 Heming Wu,4 Sandip Patil5,6 1Institute of Basic Medical Sciences, Meizhou People’s Hospital, Meizhou Academy of Medical Sciences, Meizhou, 514031, People’s Republic of China; 2Guangdong Engineering Technological Research Center of Clinical Molecular Diagnosis and Antibody Drugs, Meizhou Academy of Medical Sciences, Meizhou, 514031, People’s Republic of China; 3Institute of Cardiovascular Disease, Meizhou People’s Hospital, Meizhou Academy of Medical Sciences, Meizhou, Guangdong, 514031, People’s Republic of China; 4Meizhou Municipal Engineering and Technology Research Centre for Molecular Diagnostics of Major Genetic Disorders, Meizhou People’s Hospital, Meizhou Academy of Medical Sciences, Meizhou, 514031, People’s Republic of China; 5Department of Hematology and Oncology, Shenzhen Children’s Hospital, Shenzhen, 518038, People’s Republic of China; 6Paediatric Research Institute, Shenzhen Children’s Hospital, Shenzhen, 518038, People’s Republic of China*These authors contributed equally to this workCorrespondence: Heming Wu; Sandip Patil, Email [email protected]; [email protected]: The polymorphism of the solute carrier organic anion transporter family member 1B1 (SLCO1B1) 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 SLCO1B1 genetic variants utilizing Duplex Fluorescence Melting Curve Analysis (DFMCA).Methods: 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 SLCO1B1 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.Results: 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.Conclusion: 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.Keywords: SLCO1B1, melting curve analysis, gene polymorphism, drug delivery