A novel reporter system for temperature dependent analysis of nucleic acid amplification tests.

BackgroundReal-time polymerase chain reaction (qPCR) is a key technology in molecular diagnostics, with multiplexing further improving efficiency for pathogen detection. However, multiplexing is limited by the number of optical channels available for probe differentiation. Temperature-dependent reporter systems like TOCE and TAGS address this by introducing temperature as a second analytical dimension, expanding multiplexing potential. Here, we introduce "Target-mediated branched overlap extension" (TBOE) as a new reporter technology that enables temperature-dependent signal generation without requiring probe digestion, making it compatible with both qPCR and isothermal amplification methods. MethodsPrototype assays were designed using publicly available sequence data in conjunction with Geneious Prime and PrimerQuest software. The TBOE reporter consists of two oligonucleotide (Head and Tail oligo) that are positioned at two adjacent target sites within the amplified regions. The interaction between both oligos is facilitated by simultaneously binding to the target and a shared overlap sequence between the two oligos, thereby forming a triplex. The elongation of the head-oligo leads to duplex formation with the tag-sequence, thus generating detectable signals. Analytical experiments were carried out using clinical bacterial isolates containing carbapenemase genes or anonymized clinical remnant samples containing relevant bacterial target sequences. ResultsIn contrast to TaqMan and TOCE, the TBOE reporter generated temperature dependent signals both in conjunction with 5 exonuclease and without 5 exonuclease activity in the mastermix used. A LAMP assay was created, demonstrating the ability of the TBOE reporter to generate temperature dependent signals in the context of an isothermal amplification assay. A set of carbapenemase assays were created to demonstrate the multiplexing potential of the TBOE reporter with both analysis by melt curve analysis and detection of different temperature levels during amplification. Finally, a SNP detection assay using TBOE reporters was created and tested in a clinical sample set. DiscussionTaqMan-based multiplex qPCR has advanced pathogen detection but is limited in multiplexing capacity. We introduce Target-mediated branched overlap extension (TBOE), a novel temperature-dependent reporter system compatible with qPCR and isothermal amplification. TBOE enables high-order multiplexing without probe cleavage, expanding diagnostic applications, including SNP detection and LAMP-based assays.