Novel RecA Enzyme Kit for Rapid ASFV Nucleic Acid Detection
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Solution Overview
Problem
Current methods for detecting African swine fever virus (ASFV) have limitations such as low sensitivity, long detection times, and the potential for false negatives, especially in low viral burden samples, making them inadequate for rapid and accurate diagnosis in field settings.
Innovation Solution
A rapid viral nucleic acid detection kit utilizing a novel RecA enzyme with improved solubility and recombinase activity, combined with specific primers and proteins like uvsY and ssb, for isothermal nucleic acid amplification, allowing for sensitive and specific detection of ASFV in as little as 5 copies/μL within 5-20 minutes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR method is used for ASFV detection, then detection accuracy is high, but detection time is long and operation is complicated
Solution Approach 1:
The patent changes the fundamental parameter of the amplification method from conventional PCR (requiring temperature cycling) to isothermal amplification using RecA enzyme and ssb protein, which maintains constant temperature. This parameter change enables detection within 20-30 minutes while preserving high accuracy, directly resolving the contradiction between detection time and accuracy
Solution Approach 2:
The patent replaces the complex mechanical temperature cycling system of conventional PCR with a biochemical isothermal amplification system using RecA enzyme and ssb protein. This substitution eliminates the need for complex thermal cycler equipment and lengthy temperature cycling protocols, achieving rapid detection (20-30 minutes) with maintained accuracy
2Measurement precision
If real-time fluorescence-based quantitative PCR is used, then sensitivity and specificity are high, but equipment cost is expensive and operation requires high technical skill
Solution Approach 1:
The patent employs disposable colloidal gold test paper strips that can be discarded after single use, replacing expensive real-time fluorescence detection equipment. The test strips provide high sensitivity detection through visual color change interpretation, eliminating the need for costly instruments while maintaining detection capability
Solution Approach 2:
The patent substitutes complex electronic fluorescence detection systems with simple optical detection using colloidal gold test paper strips. The detection mechanism changes from measuring fluorescent signals requiring sophisticated equipment to visual colorimetric analysis that can be performed with minimal equipment, reducing cost while preserving sensitivity
3Ease of operation
If RAA isothermal fluorescence detection method is used, then operation is simplified, but sensitivity is low and detection time is long
Solution Approach 1:
The patent creates a composite enzymatic system combining RecA enzyme with ssb protein and T7 DNA polymerase, where each component contributes specific functions. This composite system achieves both high sensitivity (detecting 100 copies/μL) and operational simplicity, overcoming the limitations of RAA while maintaining ease of use
Solution Approach 2:
The patent introduces ssb protein as an intermediary component that enhances the binding efficiency between RecA enzyme and DNA template, thereby improving detection sensitivity. The ssb protein acts as a mediator that facilitates more effective nucleic acid amplification, achieving 100 copies/μL sensitivity while keeping the operation simple
4Speed
If existing RecA enzyme is used for isothermal amplification, then detection speed is improved, but sensitivity is insufficient due to low recombinase mediation efficiency
Solution Approach 1:
The patent optimizes the concentration parameters of RecA enzyme and ssb protein in the reaction system, finding the optimal ratio that maximizes both amplification speed and sensitivity. By adjusting these parameters, the system achieves rapid detection (20-30 minutes) with high sensitivity (100 copies/μL), resolving the contradiction between speed and sensitivity
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 significantly enhances detection sensitivity and speed, reducing the risk of missed or false detections, and is suitable for both PCR and isothermal systems, making it suitable for use in remote areas with minimal equipment requirements.
Implementation Method 1
A rapid viral nucleic acid detection kit utilizing a novel RecA enzyme with improved solubility and recombinase activity
Implementation Method 2
for isothermal nucleic acid amplification, allowing for sensitive and specific detection of ASFV
Implementation Method 3
combined with specific primers and proteins like uvsY and ssb, for isothermal nucleic acid amplification
Data Source
AI summary
The present invention provides a rapid viral nucleic acid detection kit prepared by using a novel RecA enzyme and the detection method thereof. By editing the recombinase RecA gene, the expressed RecA protein has better solubility and recombinase activity. The RecA protein is used to prepare recombinase dry powder, further the formula of the recombinase dry powder and the ratio are optimized; and specific primers for the ASFV p72 gene are designed for the rapid nucleic acid detection of ASFV, significantly improving the detection sensitivity. In addition, the detection time is short, which effectively avoids missed detection and false detection, and helps prevention of epidemic.