Isothermal PCR Calibration Kit Design
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Solution Overview
Problem
The lack of calibration technology and standard kits for isothermal PCR instruments hinders the development of calibration work, as existing methods and kits for real-time fluorescence quantitative PCR instruments are not suitable for isothermal PCR instruments due to differences in primer-probe systems, amplification enzyme reactions, and temperature requirements.
Innovation Solution
A dedicated kit for calibrating isothermal PCR instruments is developed, including a primer set with specific nucleotide sequences, a standard DNA plasmid, reaction buffer, polymerase, and dye, optimized for loop-mediated isothermal amplification (LAMP) technology to ensure accurate calibration and performance evaluation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If calibration methods and kits for real-time fluorescence quantitative PCR instruments are used for isothermal PCR instruments, then calibration work can be conducted, but the calibration accuracy and suitability are compromised due to differences in primer-probe systems, amplification enzyme reactions, and temperature requirements
Solution Approach 1:
The patent changes the fundamental parameters of the calibration system by using isothermal amplification instead of thermal cycling, designing primers specific to LAMP reactions, and establishing calibration curves at constant temperature (e.g., 65°C) rather than through temperature cycles. This transforms the calibration method to match the specific operational parameters of isothermal PCR instruments.
Solution Approach 2:
The patent introduces a specialized calibration kit containing LAMP-specific primers, isothermal amplification enzymes, and calibrated standard substances as intermediaries that bridge the gap between the calibration reference and the isothermal PCR instrument. These intermediaries are specifically designed to work with the constant temperature amplification process, enabling accurate calibration without using conventional PCR reagents.
2Measurement precision
If a dedicated calibration kit for isothermal PCR instruments is developed, then calibration accuracy and suitability are improved, but the complexity of the calibration system increases due to the need for special primer-probe systems and enzyme reaction systems
Solution Approach 1:
The patent designs the calibration kit to be universally applicable to different isothermal PCR instruments and amplification systems (LAMP, NASBA, RCA, RPA) by establishing general calibration principles and using standardized calibration substances. The kit provides a universal calibration framework that can accommodate various isothermal amplification methods while maintaining consistency in calibration procedures.
Solution Approach 2:
The patent segments the calibration system into distinct modular components: calibration substances with known concentrations, LAMP-specific primer sets, reaction buffers, and amplification enzymes. This segmentation allows each component to be independently optimized and validated, making the overall system more manageable and easier to implement despite the increased complexity.
3Productivity
If isothermal PCR instruments are designed for constant temperature operation, then reaction efficiency and simplicity are improved, but the difficulty of calibration increases due to the absence of temperature-changing processes
Solution Approach 1:
The patent replaces the thermal cycling mechanism (heating and cooling cycles) with a constant temperature amplification system. Instead of relying on temperature changes to drive the amplification process, the system uses isothermal conditions with specialized enzymes that function optimally at a single temperature. This substitution simplifies the instrument operation but requires a corresponding substitution in the calibration approach, using constant temperature calibration curves instead of temperature-dependent calibration.
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
The kit provides a comprehensive calibration method applicable to all isothermal PCR instruments, ensuring accurate and reliable nucleic acid detection results by optimizing primer design, reaction conditions, and temperature settings, thus supporting the normal operation of isothermal PCR instruments across various amplification systems.
Implementation Method 1
a primer set with specific nucleotide sequences
Implementation Method 2
polymerase, and a dye, optimized for loop-mediated isothermal amplification (LAMP) technology
Implementation Method 3
a primer set with specific nucleotide sequences, a standard DNA plasmid, reaction buffer, polymerase, and dye
Data Source
AI summary
Provided is a kit for calibration of an isothermal polymerase chain reaction (PCR) analyzer and use thereof. The kit includes a standard substance, an amplification primer set, a reaction buffer, a polymerase, a dye, and a negative control. The standard substance is a DNA plasmid with a gradient concentration of 100 copies/μL to 106 copies/μL; the amplification primer set has nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6; the polymerase is a Bst DNA polymerase; and the dye is a loop-mediated isothermal amplification (LAMP) fluorescent dye (with excitation: 485 nm, emission: 498 nm, and a detection channel of SYBR® Green I or a FAM channel). The kit and a technology for calibration of an isothermal PCR instrument meet calibration demands of the isothermal PCR instrument and fill a technical gap.