PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• Current PCR-based diagnostics are limited by the number of targets detectable per fluorescent channel.
• Multiplexing assays often require multiple channels, increasing complexity and cost.
• There is a need for enhanced multiplexing capabilities in PCR to improve diagnostic efficiency.

Purpose of the Study:

• To introduce and evaluate PlexProbes, a novel probe technology for temperature-dependent, single-channel multiplex PCR.
• To demonstrate the ability of PlexProbes to discriminate between multiple targets at different temperatures within a single fluorescent channel.
• To assess the performance of a PlexProbe-based multiplex assay for sexually transmitted infections (STI) using clinical samples.

Main Methods:

• Development of temperature-responsive "on/off" fluorescent probes (PlexProbes).
• Design of a model duplex assay for simultaneous detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) at distinct temperatures (52°C and 74°C).
• Construction and validation of a multiplex PlexProbe assay for six STI targets (GC, CT, Mycoplasma genitalium, Trichomonas vaginalis, and internal control) using three fluorescent channels.

Main Results:

• The model duplex assay successfully detected as few as 40 copies of CT and GC, individually or in co-infection, in a single channel.
• The PlexProbe prototype assay for STI (PP-STI) simultaneously detected six targets.
• The PP-STI assay demonstrated >99.5% overall agreement with a commercial reference assay on 337 clinical samples.

Conclusions:

• PlexProbes technology enables enhanced multiplexing in PCR by allowing target discrimination within a single fluorescent channel based on temperature.
• This approach effectively doubles the number of targets detectable on existing PCR platforms.
• The PP-STI assay showcases the clinical utility of PlexProbes for efficient, cost-effective, and accurate multi-target infectious disease diagnostics.