RCL Detection Reagent Primer Probe Optimization
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Solution Overview
Problem
Current primers and probes for detecting Replication Competent Lentivirus (RCL) by fluorescence quantitative real-time PCR have low amplification efficiency and poor specificity, necessitating the development of new reagents and methods for effective detection.
Innovation Solution
A reagent combination comprising specific primer pairs and probes for amplifying the VSV-G gene of RCL, optimized for high amplification efficiency and specificity, is used in conjunction with the TaqMan probe method for fluorescence quantitative real-time PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current primers and probes are used for detecting RCL by fluorescence quantitative real-time PCR, then the detection can be performed, but the amplification efficiency is low and specificity is poor
Solution Approach 1:
The patent optimizes the nucleotide sequences, lengths, and compositions of primers and probes to improve amplification efficiency and specificity. By changing the parameters of the detection reagents (primer/probe sequences and structures), the patent achieves both high reliability and high productivity in RCL detection
Solution Approach 2:
The patent uses a composite detection system combining multiple primers and probes with different sequences and specificities. This composite approach allows simultaneous detection of multiple RCL variants while maintaining high amplification efficiency and specificity for each target
2Reliability
If standard cell co-cultivation method is used for detecting RCL, then the detection is performed, but the detection cycle is long taking about 6 weeks or longer
Solution Approach 1:
The patent replaces the mechanical/biological cell co-cultivation system with a molecular biology-based fluorescence quantitative real-time PCR system. This substitution dramatically reduces detection time from 6 weeks to several hours while maintaining detection reliability through specific primer/probe design
Solution Approach 2:
The patent performs DNA extraction and amplification in advance, allowing for rapid detection results. By preparing the detection reagents and performing preliminary amplification steps, the patent eliminates the lengthy waiting period required for cell culture methods
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 proposed solution achieves high amplification efficiency (≥90%) and good specificity, making it suitable for clinical and laboratory detection of RCL, thereby addressing the limitations of existing detection methods.
Implementation Method 1
The core is to use the 3′→5′ exonuclease activity of Taq enzyme to cut off the probe to generate a fluorescent signal
Implementation Method 2
cut off the probe to generate a fluorescent signal
Implementation Method 3
the probe and the template are specifically bound, the intensity of the fluorescence signal represents the quantity of the templates
Data Source
AI summary
The present invention provides a reagent and method for detecting a replication-competent lentivirus (RCL) by fluorescence quantitative real-time polymerase chain reaction (PCR). In particular, the present invention provides a primer and probe combination for detecting RCL, and a method for performing detection using said primer and probe; the present invention also provides a reagent kit comprising said primer and probe. The primer and probe combination of the present invention detects RCL with high amplification efficiency and good specificity, and can be used for RCL detection and RCL monitoring of clinical patient peripheral blood samples which may occur during a production process.