A probe-based higher order multiplex technology to expand the capacity of digital PCR systems

Abstract Absolute quantification of a DNA or RNA biomarkers of interest is needed in many biomedical applications. Digital PCR (dPCR) provides ultrasensitive and absolute nucleic acid quantification of DNA or RNA biomarker of interest and is becoming a popular tool with wider utility, especially, where absolute quantification is essential. Some of the dPCR applications are detection of rare allele, measurement of copy number variation and gene fusions, viral titer measurement, quantification of next-generation sequencing libraries, and detection of rare targets from environmental samples such as wastewater. Currently available dPCR systems are limited by the availability of optical channels and comes with high cost; instrument and consumables, and thus, has a negative effect on clinical and hospital laboratory operation. Therefore, there is a need for a reliable higher order multiplexing method that decreases the cost, maximizes the capacity, decreases the lab operational burden, requires small quantities of precious clinical samples, and reduces the environmental waste (decrease in environmental waste from reduction in plastics and chemical reagents). Currently, higher order multiplexing in digital PCR is achieved by amplitude modulation, requiring a complex data analysis tool, thorough optimization of probe concentration, and multiple controls. Our Zip-MeltTM Multiplex Probe Technology (Zip-MeltTM MPT) - a probe-based higher order multiplexing method, provides a simple alternative solution. The goal of this Phase 1 application is to develop high multiplex digital PCR method for detection of important gene fusions using innovative Zip-MeltTM MPT. To demonstrate the feasibility, herein, we chose quantitative detection and discrimination of four BCR-ABL1 fusion gene variants important for monitoring of chronic myeloid leukemia utilizing just one optical channel, followed by performance characteristics studies in comparison to the Comparator Method. The proposed studies will demonstrate and mature our proprietary probe-based simple multiplex platform for quantitative detection of multianalytes in a single dPCR reaction/well to measure residual diseases for diagnosis or monitoring of cancer.