B-233 A universal probe system for maximising real-time qPCR multiplex capacity: detection of multiple targets per fluorescent channel

Abstract Background Recently, a novel single-channel multiplex qPCR probe system was developed to allow real-time detection of three or four targets within a single fluorescent channel. A major limitation for multiplex qPCR is the 4-6 fluorescent channels in most qPCR instruments, with most technologies only allowing detection of 1 target per channel. This system employs multiple probes, each designed to emit fluorescence at a distinct temperature within a single optical channel, but only when its corresponding target is present. The fluorescence signals from the probes are confined to their specific temperature ranges, with minimal to no cross talk between them. Each probe is designed to be universal, making the system versatile and adaptable to many applications. Model multiplex qPCR assays were developed to demonstrate the use of the technology. The qPCR cycling protocols included either 3 or 4 fluorescence acquisition temperatures. The first assay was designed to detect Neisseria gonorrhoeae (NG) at the first temperature (45°C), Chlamydia trachomatis (CT) at the second temperature (61°C), and Mycoplasma genitalium (MG) at the third temperature (78°C), all in the FAM channel. The second assay was designed to detect NG at the first temperature (34°C), MG at the second temperature (56°C), CT at the third temperature (70°C) and Trichomonas vaginalis (TV) at the fourth temperature (82°C), all within the FAM channel. Methods The qPCR cycling on the CFX96™ Touch Real-Time PCR Detection System (Bio-Rad) was programmed to acquire fluorescence at three or four acquisition temperatures during amplification cycling. Synthetic templates of all targets were tested in two replicates at high and low quantities as well as mixed in equal amounts to simulate scenarios of co-infections. Results The first model assay was developed for detection of 3 bacterial targets in a single fluorescent channel. The development of a second assay followed, for detection of 4 targets in a single fluorescent channel. Both assays demonstrated minimal cross talk between the different acquisition temperatures. Detection of single and mixed targets showed comparable performance with both assays. Conclusion The novel universal multiplex qPCR detection system is a highly versatile and sensitive tool, enabling detection of 3 or 4 targets independently in a single fluorescent channel. This technology greatly increases the multiplexing capacity of qPCR; on a 6-channel instrument, it is theoretically possible to detect up to 24 targets with a single test. On the other hand, this technology can greatly increase multiplexing capacity on instruments with limited number of channels e.g. point-of-care qPCR instruments. The ease of adaptability combined with high multiplexing capacity makes this technology suitable to various applications from human diagnostics to food microbiology.