Unlike conventional multiplex PCR methods that rely on limiting primer concentrations or suffer from cold-start artifacts, this approach kinetically separates each amplification reaction via temperature staging rather than reagent titration. The hot-start RT step minimizes background priming, while the rapid-cycle design drastically reduces assay time. By obviating primer limiting and incorporating two distinct thermal windows, this method offers superior sensitivity, reproducibility, and ease of use for clinical and intraoperative molecular diagnostics.
Description
This technology integrates reverse transcription and PCR amplification in a single tube by applying two or more sequential thermal stages with distinct annealing/extension temperatures and cycle parameters. An initial high-temperature stage preferentially amplifies one target amplicon, followed by a lower-temperature stage that drives production of a second amplicon, achieving balanced output without adjusting primer concentrations. The reverse transcription step is performed as a two-minute hot-start reaction at elevated temperature to suppress nonspecific priming, and rapid thermal cycling (1-second denaturation, 6-second extension over 40 cycles) completes quantitative RT-PCR in under twenty minutes. These optimized conditions boost sensitivity for low-abundance transcripts and enable simultaneous detection of an endogenous control and a diagnostic marker (e.g., carcinoembryonic antigen) in a single-tube, intraoperative assay.
Applications
Intraoperative cancer metastasis detection
Point-of-care infectious disease testing
Multiplex viral panel diagnostics
Personalized oncology biomarker monitoring
Veterinary pathogen rapid diagnostics
Advantages
Balanced multiplex amplification without primer titration
Rapid quantitative RT-PCR workflow completed in under 20 minutes
Suppression of nonspecific priming for improved assay specificity
Enhanced sensitivity for low-abundance and rare targets
Single-tube format that simplifies workflow and reduces contamination risk
Simultaneous detection of endogenous controls and diagnostic transcripts
Enables intraoperative micrometastasis detection for real-time surgical decision making
Invention Readiness
A single-tube RT-PCR uses two sequential thermal stages with distinct anneal/extension temperatures to kinetically separate multiplex target amplification. A high-temperature phase favors one amplicon, then a lower-temperature phase promotes a second, balancing yields without primer titration. A two-minute hot-start reverse transcription at elevated temperature suppresses nonspecific priming, followed by 40 rapid cycles (1 s denaturation/6 s extension), enabling sensitive multiplex detection of endogenous controls and diagnostic transcripts in under 20 minutes.
IP Status
Abandoned -
https://patents.google.com/patent/US20060205006A1
