Microsatellite Instability Detection Kit Using Negative Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting microsatellite instability face challenges in accuracy and sensitivity, particularly due to contamination risks and the need for normal tissue controls, which complicates the detection process and may lead to aerosol pollution.
Innovation Solution
A kit and method utilizing qPCR reaction solutions with specific primers and probes for detecting microsatellite loci NR-21, NR-24, NR-27, MONO-27, BAT-25, and BAT-26, along with a negative control and Premix Ex Taq, that calculates ΔΔCt values to determine MSI or MSS without requiring normal tissue controls, thereby reducing contamination risks and ensuring high sensitivity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If normal tissue is used as control for MSI detection, then detection reference is improved, but contamination risk increases due to exposure and aerosol pollution
Solution Approach 1:
The patent extracts the control function from external normal tissue samples and embeds it within the detection kit itself through the negative control solution containing SW480 cell line DNA. This eliminates the need to handle separate normal tissue samples, removing the source of aerosol contamination while maintaining the reference function for accurate MSI detection.
Solution Approach 2:
The patent introduces a synthetic negative control solution as an intermediary that replaces the need for actual normal tissue controls. This intermediary contains predetermined DNA sequences that serve as reference points for detection without requiring exposure to external biological samples, thus preventing contamination while providing detection reference.
2Measurement precision
If multiple detection steps and normal tissue controls are used, then detection accuracy is improved, but operation complexity increases
Solution Approach 1:
The patent merges the control sample preparation, reference standard, and detection reagents into a single integrated qPCR detection kit. The negative control solution is pre-prepared with known DNA sequences, eliminating multiple separate preparation steps and simplifying the overall detection process while maintaining accuracy through built-in reference points.
Solution Approach 2:
The patent performs preliminary preparation of the negative control solution with predetermined DNA concentrations and sequences before the detection process. This pre-prepared control is included in the kit, eliminating the need for complex real-time control preparation during detection and reducing operational steps while ensuring consistent reference standards.
3Measurement precision
If qPCR reaction solutions with specific probes are used, then detection sensitivity is improved, but reagent complexity increases
Solution Approach 1:
The patent designs qPCR reaction solutions with probes that can detect multiple microsatellite loci (NR-21, NR-24, NR-27, BAT-25, BAT-26) using the same basic reagent system. The universal qPCR premix and probe design allow detection of different loci without requiring separate complex reagent preparations, reducing overall reagent complexity while maintaining high sensitivity through specific probe-labeled nucleotide sequences.
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 solution provides a simple, accurate, and sensitive method for detecting microsatellite instability in tissues or ctDNA, avoiding aerosol pollution and ensuring consistent amplification, with clear criteria for determining MSI or MSS based on ΔΔCt values, thus improving detection precision and operational ease.
Implementation Method 1
a kit for detecting microsatellite instability is provided. The kit includes a negative control, a plurality of qPCR reaction solutions, a qPCR premixes and a sterile enzyme-free water
Implementation Method 2
the reference probe shown in SEQ ID No: 3, and the detection probe shown in SEQ ID No: 4; wherein, the nucleotide sequence of SEQ ID No: 3 has a VIC label at its 5′ end, and a MGB label at its 3′ end; and furthermore, the nucleotide sequence of SEQ ID No: 4 has a FAM label at its 5′ end, and has a MGB label at 3′ end
Data Source
AI summary
The disclosure provides a kit for detecting microsatellite instability and a method therefor. The kit includes a negative control, a plurality of qPCR reaction solutions, a qPCR premix and a sterile enzyme-free water; the plurality of qPCR reaction solutions includes 6 pairs of upstream primers and downstream primers of which the MSI mutation site is amplified, and a reference probe for the internal reference and a detection probe for the mutation site. The difference between the amplification of the gene and the gene at the mutation site of the samples and the negative control is used to detect the microsatellite instability. The method and kit as provided is easy and simple without the need of normal tissues being a control, and the need to open the cap. By doing so, aerosol pollution is avoided and sample supplies are conserved.