Rationally\nDesigned Universal Melting Probes (Uni-Melt)\nfor Multiplex Genotyping

Polymerase chain reaction (PCR) with probe-based melting\ncurve\ntechniques has been widely used in multiplex genotyping for early\ndiagnosis of genetic diseases and common cancers. However, traditional\ndesigns face limitations due to the high modification cost of sensing\nprobes and complex background fluorescence signals. Herein, we introduce\nthe Universal Melting Probes (Uni-Melt) system, a closed-tube PCR\nassay for multiplex genotyping that incorporates barcoded PCR amplification\nand universal hybridization probes for melting curve analysis. Uni-Melt\ncan detect multiple genotypes using a single set of universal fluorophore-modified\nprobes and unmodified target-specific mediator strands, which was\nverified by detecting five different human papillomavirus (HPV) genotypes\nutilizing two fluorescent channels with the lowest detectable concentration\nat 10<sup>2</sup> copies/μL. In addition, Uni-Melt can differentiate\nmultiple mutation sites including single nucleotide polymorphisms\n(SNPs), as demonstrated in the SLC25A13 gene. Overall, Uni-Melt is\na closed-tube, sensitive, and specific system for multiplexed genotyping\ncompatible with commonly employed quantitative polymerase chain reaction\n(qPCR) thermocyclers. Compared to the traditional probe-based melting\ncurve analysis, Uni-Melt can reduce the assay cost associated with\nmultiple probe modifications and decrease the complexity of the reaction\nwithout sacrificing sensitivity and specificity, which makes it a\npractically useful tool for clinical screening.