Fluorescent Probe Primer Pair for sgRNA Residue Detection
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Solution Overview
Problem
Current methods for detecting residual small guide RNA (sgRNA) in environments are not specific and can lead to off-target effects due to cross-contamination, with traditional methods like TOC determination being non-specific and Next-Generation Sequencing (NGS) being costly and time-consuming.
Innovation Solution
A primer pair and kit using a fluorescent probe for real-time fluorescence quantitative PCR (RT-qPCR) are designed to specifically detect residual sgRNA, with the primer pair and probe sequences provided, allowing for accurate and efficient detection of sgRNA residues in environmental samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional residue detection methods like TOC determination are used, then the detection process is simple, but the detection is not specific and cannot directly reflect sgRNA residue
Solution Approach 1:
The patent introduces a fluorescent probe as an intermediary molecule that specifically binds to sgRNA sequences. This probe acts as a mediator between the detection system and the target sgRNA, enabling specific detection through fluorescence signal changes when the probe hybridizes with the target sequence, thus resolving the contradiction between detection simplicity and specificity.
Solution Approach 2:
The patent replaces traditional mechanical/chemical detection methods (like TOC determination) with a biological recognition system based on nucleic acid hybridization and fluorescence detection. This substitution enables sequence-specific detection by utilizing the complementary base pairing between the fluorescent probe and sgRNA, achieving high measurement precision without excessive complexity.
2Measurement precision
If NGS sequencing method is used for sgRNA residue detection, then the detection is theoretically feasible, but the experimental cycle is long and costs are high
Solution Approach 1:
The patent extracts only the essential detection function from the complex NGS sequencing process by using a targeted fluorescent probe approach. Instead of sequencing the entire genome or all RNA molecules, the method specifically targets and detects sgRNA sequences using a designed fluorescent probe, thereby achieving accurate detection with significantly reduced time and cost while maintaining measurement precision.
Solution Approach 2:
The patent applies partial action by focusing detection efforts only on the specific sgRNA sequences of interest rather than performing comprehensive sequencing. The fluorescent probe is designed to hybridize with specific regions of the sgRNA, enabling targeted detection that achieves sufficient accuracy for quality control purposes without the time and resource expenditure of full NGS sequencing.
3Measurement precision
If NGS sequencing method is used for sgRNA residue detection, then the detection is theoretically feasible, but the costs are high
Solution Approach 1:
The patent employs a cost-effective fluorescent probe that can be synthesized at low cost compared to NGS sequencing reagents and infrastructure. The probe is a short oligonucleotide sequence designed to specifically bind sgRNA, and it can be used in a disposable qPCR format, eliminating the need for expensive NGS equipment and complex data analysis pipelines while maintaining detection accuracy.
Solution Approach 2:
The patent substitutes the expensive and complex NGS sequencing system with a simpler and more economical fluorescent-based detection system. By using fluorescence qPCR with a specifically designed probe, the method achieves comparable or sufficient detection accuracy at a fraction of the cost, making it suitable for routine quality control and environmental monitoring of sgRNA residues.
4Productivity
If cross-contamination occurs during collinear production, then the production efficiency is maintained, but the quality of next batch products is affected
Solution Approach 1:
The patent implements preliminary detection of sgRNA residues in the production environment before next batch production begins. By using the fluorescent probe-based qPCR method to screen for residual sgRNA on equipment and in the environment, the system can identify contamination risks in advance, allowing for cleaning validation and preventive measures to be taken before quality issues affect the next batch, thus maintaining both productivity and reliability.
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 method provides specific, sensitive, and repeatable detection of sgRNA residues, reducing the risk of cross-contamination and ensuring compliance with cleaning validation standards, with results showing significant reduction in residues post-clearance and minimal impact on downstream biological applications.
Implementation Method 1
a fluorescent probe, the fluorescent probe is a nucleic acid molecule as shown in SEQ ID NO: 4 with a fluorescent group connected at 5′-terminal of the nucleic acid molecule and a quenching group connected at 3′-terminal of the nucleic acid molecule
Implementation Method 2
performing real-time fluorescence quantitative PCR on the sample obtained from the local sampling using the primer pair and the probe in step 1)
Data Source
AI summary
A primer pair, a kit and a method for detecting the residual amount of sgRNA in the environment. The forward primer of the primer pair is a nucleic acid molecule as shown in SEQ ID NO: 2, and the reverse primer of the primer pair is a nucleic acid molecule as shown in SEQ ID NO: 5. The primer pair can be used for detecting the residual amount of sgRNA in the environment; can perform real-time and high-throughput detection on the residual amount of sgRNA in the environment; has the advantages of good specificity, high sensitivity and repeatability, and convenient operation; and can perform real-time and high-throughput detection on the residual amount of the sgRNA in the environment in different stages of a production process.