CSFV Detection via NS5A Region Specific Primers
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Solution Overview
Problem
Current methods for detecting Classical Swine Fever Virus (CSFV) face challenges with amplicon contamination and interference from other RT-PCR applications, necessitating a specific and reliable detection method that is compatible with reference gene detection and can differentiate CSFV from closely related viruses like BVDV and BDV.
Innovation Solution
The method involves detecting the NS5A region of the CSFV genome using specific primers and probes, with a focus on the 3'-end nucleotides, and utilizing nucleic acid amplification techniques like real-time RT-PCR to identify CSFV, even in samples with high throughput of positive materials, ensuring specificity and compatibility with reference gene detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RT-PCR methods are used for CSFV detection, then detection sensitivity is improved, but false positives from related viruses (BVDV, BDV) and amplicon contamination occur
Solution Approach 1:
The patent applies local quality by designing primers and probes that target a specific local region (NS5A gene) within the CSFV genome. The forward primer binds to nucleotides 9388-9407 and the reverse primer to 9468-9487 of the NS5A region, creating highly specific local recognition sequences that differentiate CSFV from related viruses while maintaining high detection sensitivity
Solution Approach 2:
The patent segments the detection system into three distinct functional components: a forward primer (nucleotides 9388-9407), a reverse primer (nucleotides 9468-9487), and a fluorescent probe targeting the intervening NS5A region. This segmentation allows each component to be optimized independently for specificity and sensitivity, resolving the contradiction between detection precision and reliability
2Productivity
If multiple RT-PCR applications are run in parallel, then productivity is improved, but interference between applications increases
Solution Approach 1:
The patent uses a fluorescent probe as an intermediary detection mechanism that emits signals at specific wavelengths unique to each RT-PCR application. This allows multiple applications to be run in parallel on the same instrument without cross-interference, as each reaction can be monitored independently through its distinctive fluorescent signature, thereby maintaining both high 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
This approach allows for the sensitive and specific detection of all CSFV strains, including those with unknown NS5A regions, while avoiding false positives from related viruses, thereby improving diagnostic accuracy and reliability in high-throughput settings.
Implementation Method 1
detection of the NS5A region of at least one classical swine fewer virus (CSFV) genome in a sample derived from said subject; by means of nucleic acid amplification
Data Source
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AI summary
The present invention relates to a method for determining the presence of a classical swine fewer virus (CSFV) in a subject comprising the detection of the NS5A region of at least one classical swine fewer virus (CSFV) genome in a sample derived from said subject. The invention further relates to a kit for determining the presence of a classical swine fewer virus (CSFV) and to nucleic acids as well as to the use of the kit and/or nucleic acids in said method.