Rationally Designed Universal Melting Probes (Uni-Melt) for Multiplex Genotyping

Polymerase chain reaction (PCR) with probe-based melting curve techniques has been widely used in multiplex genotyping for early diagnosis of genetic diseases and common cancers. However, traditional designs face limitations due to the high modification cost of sensing probes and complex background fluorescence signals. Herein, we introduce the Universal Melting Probes (Uni-Melt) system, a closed-tube PCR assay for multiplex genotyping that incorporates barcoded PCR amplification and universal hybridization probes for melting curve analysis. Uni-Melt can detect multiple genotypes using a single set of universal fluorophore-modified probes and unmodified target-specific mediator strands, which was verified by detecting five different human papillomavirus (HPV) genotypes utilizing two fluorescent channels with the lowest detectable concentration at 10² copies/μL. In addition, Uni-Melt can differentiate multiple mutation sites including single nucleotide polymorphisms (SNPs), as demonstrated in the SLC25A13 gene. Overall, Uni-Melt is a closed-tube, sensitive, and specific system for multiplexed genotyping compatible with commonly employed quantitative polymerase chain reaction (qPCR) thermocyclers. Compared to the traditional probe-based melting curve analysis, Uni-Melt can reduce the assay cost associated with multiple probe modifications and decrease the complexity of the reaction without sacrificing sensitivity and specificity, which makes it a practically useful tool for clinical screening.