An Efficient Pharmacogenomic Assay for Psychotropic Drugs by Multiplex Fluorescence Melting Curve Analysis.

Long-term pharmacotherapy is the primary approach for alleviating the symptoms of mental disorders. Numerous pharmacogenomic studies have confirmed that genetic polymorphisms are key factors underlying inter-ethnic and inter-individual differences in drug response. Pharmacogenomic testing is an essential pathway to achieve precision medicine. In this study, seven common single nucleotide polymorphisms (SNPs) closely associated with the efficacy of antidepressants, and antipsychotics were screened and identified through comprehensive literature retrieval and mining of authoritative databases. These SNPs are located in the ANKK1, DRD2, FKBP5, GRIK4, HTR1A, HTR2A, and MC4R genes. Utilizing multiplex fluorescence melting curve analysis (mFMCA), a four-channel multiplex fluorescent PCR system was successfully established, enabling the simultaneous genotyping of 14 distinct alleles across 7 SNP loci in a single tube. Analytical performance including sensitivity and specificity as well as reproducibility was rigorously evaluated. Specifically, a two phase validation was conducted. An initial calibration phase using 98 clinical samples within a training cohort was used to establish integrative genotyping criteria. These criteria combined statistical melting temperature (T m) intervals, defined as the Mean T m±3 standard deviations (SDs), with peak morphology. The established criteria were then evaluated in a blinded validation phase using an independent cohort of 124 clinical samples. The mFMCA system can accurately detect genomic DNA at concentrations as low as 100 copies/μL, and exhibits high specificity and no cross-reactivity. In the validation cohort (n=124), the mFMCA-derived genotypes achieved 100% concordance with Sanger sequencing. Furthermore, the high stability of the system was further evidenced by its consistent performance across varying DNA concentrations, different clinical samples, and multiple instruments. This study successfully established a dedicated and cost-effective mFMCA assay for psychotropic pharmacogenomic testing. The method provides an efficient, accurate, rapid, economical tool for personalized medicine in psychiatric care.