A Novel Mediator Probe and Universal Fluorescence Probe (MP-UP) System for Highly Sensitive and Versatile Nucleic Acid Detection

Abstract

Real-time qPCR and digital PCR are powerful techniques for detecting nucleic acids with high specificity and sensitivity, relying on sequence-specific labeled probes. However, each target requires the design and synthesis of specific probes, incurring significant costs. Therefore, the development of a specific and universal detection technique is both urgent and beneficial. Previously, a universal detection method was developed using a modified mediator probe and a universal hairpin reporter. However, our experiments uncovered that the mediator primers, generated by Taq polymerase cleaving mediator probes, deviate from the expected sequence, indicating a severe compromise in detection ability. Additionally, the high cost of the modified mediator probe and hairpin reporter impedes the practicality of their method. To overcome these challenges, we have developed a novel universal detection method (MP-UP) using Taqman probes, unmodified mediator probes and helper target, enabling the utilization of all mediator primers to produce abundant fluorescence. Enhanced MP-UP qPCR with an increased signal-to-noise ratio, MP-UP ddPCR, and multiplex MP-UP were established, exhibiting remarkable sensitivity, specificity, and precision comparable to specific probe assays or commercial testing kit. The mediator probe and helper target cost only one-tenth of a specific Taqman probe, rendering it feasible to replace the latter in specific nucleic acid detection.