Telomerase Assay Primer Concentration Optimization for Low-Cell Detection
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Solution Overview
Problem
Current methods for detecting telomerase activity in aqueous solutions are unable to accurately determine the presence of cancer cells when the solution contains only two cells, leading to false-negative results.
Innovation Solution
A method involving the use of a TS primer and a reverse primer in specific concentrations, along with a probe RNA and RNaseH enzyme, to amplify and detect telomerase reaction products, allowing for the determination of cancer cells even at low concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional telomerase activity measurement methods are used, then the detection process is simple, but the sensitivity is insufficient to detect only two cancer cells
Solution Approach 1:
The assay is divided into distinct functional modules: telomerase extension reaction, magnetic bead immobilization, PCR amplification with optimized primers, and fluorescent detection. Each module performs a specific function that contributes to overall sensitivity, allowing the complex detection process to be managed systematically while achieving detection of only two cancer cells
Solution Approach 2:
The invention transitions from direct detection to amplified detection by incorporating PCR amplification as an intermediate step. This dimensional change in the detection approach allows signal amplification from the telomerase reaction products, enabling detection of extremely low cancer cell numbers that would be invisible in direct assays
2Reliability
If PCR amplification is performed with standard primer concentrations, then the procedure is straightforward, but false-negative results occur in samples with low cancer cell content
Solution Approach 1:
The invention optimizes specific parameters of the PCR reaction: TS primer concentration (0.1-1 μM, lower than conventional), reverse primer concentration (0.02-0.06 μM), and annealing temperature (50-60°C). These parameter changes prevent primer-dimer formation and non-specific amplification while maintaining sensitivity for detecting low cancer cell loads, eliminating false-negative results
Solution Approach 2:
Magnetic beads serve as an intermediary to immobilize telomerase reaction products and facilitate their transfer to the PCR reaction. This intermediary step concentrates the reaction products and removes potential inhibitors, improving the reliability of subsequent PCR amplification in low-cell samples
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method accurately detects the presence of cancer cells in aqueous solutions, including those with only two cancer cells, by enhancing the sensitivity of telomerase activity measurement.
Implementation Method 1
activity of telomerase contained in a biological sample is measured
Implementation Method 2
The telomerase reaction products are immobilized on magnetic beads
Implementation Method 3
A DNA sequence contained in the telomerase reaction products is amplified by a PCR method
Implementation Method 4
mixing a probe RNA and an RNaseH enzyme with the PCR products obtained in the step (e) to prepare an RNaseH reaction solution
Implementation Method 5
one end and the other end of the probe RNA are modified with the fluorescent group and the quenching group, respectively; and the quenching group is capable of absorbing fluorescence from the fluorescent group
Data Source
AI summary
The present invention provides a method for determining whether or not an aqueous solution contains two or more cancer cells. The present method is characterized by the following three matters. First, the PCR solution contains the TS primer at a concentration of not less than 0.1 μM and not more than 1 μM in the present invention. Second, the PCR solution contains an ACX reverse primer. Third, the PCR solution contains the ACX reverse primer at a concentration of not less than 0.02 μM and not more than 0.06 μM in the present invention. In the present method, it is determined that an aqueous solution contains cancer cells even if the aqueous solution contains only two cancer cells.