Universal Eukaryotic DNA Amplification Control via 28S rRNA Targeting
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Solution Overview
Problem
Current molecular diagnostics lack a universal internal control for reliably detecting nucleic acids across a wide range of eukaryotic organisms, particularly for assessing sample integrity and absence of PCR inhibitors in quantitative PCR assays, with existing solutions being species-specific or costly.
Innovation Solution
A primer pair and probe set targeting the highly conserved 28S rRNA gene sequence (SEQ ID NO: 5), which allows for reliable amplification and detection across various eukaryotic organisms, including fungi, plants, arthropods, and mammals, providing a cost-effective means to verify sample integrity and absence of PCR inhibitors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If species-specific internal controls are used, then detection reliability for specific organisms is improved, but adaptability to detect across multiple eukaryotic species deteriorates
Solution Approach 1:
The patent applies universality by designing a primer pair targeting the 28S rRNA gene that functions across multiple eukaryotic species (plants, insects, mammals, fungi). The 28S rRNA gene was selected because it is highly conserved across eukaryotes, allowing a single primer set to serve as a universal internal control for diverse sample types, thereby resolving the contradiction between species-specific reliability and cross-species adaptability
2Adaptability or versatility
If universal primers for long DNA stretches are designed, then phylogenetic analysis capability is improved, but suitability for quantitative PCR deteriorates due to product length
Solution Approach 1:
The patent applies local quality by selecting a specific localized region within the 28S rRNA gene (nucleotides 1-84) that is both highly conserved across species and produces an appropriately short amplification product for qPCR. This localized selection allows the primer pair to maintain universality while meeting the length requirements for quantitative PCR applications
3Ease of operation
If commercially available primer/probe sets are used, then ease of operation is improved, but cost effectiveness deteriorates due to purchasing requirements
Solution Approach 1:
The patent applies copying by providing the complete nucleotide sequences (SEQ ID NO: 1-5) of the primer pair and probe set, enabling researchers to obtain identical reagents through synthetic oligonucleotide production rather than purchasing commercial kits. This approach maintains the ease of operation with ready-to-use sequences while dramatically improving cost effectiveness for laboratories performing multiple assays
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 universal control system enables reliable detection and quantification of nucleic acids in both qualitative and quantitative assays, ensuring accurate results across multiple species, including those from plants and insects, while being cost-effective and applicable in single- or multiplex detection formats.
Implementation Method 1
A primer pair and probe set targeting the highly conserved 28S rRNA gene sequence (SEQ ID NO: 5), which allows for reliable amplification and detection across various eukaryotic organisms
Implementation Method 2
methods involving the amplification and the detection/quantification of a universal control nucleotide sequence
Data Source
AI summary
Oligonucleotides may be universal primers and probes. Method may use these oligonucleotides for detecting or detecting and quantifying a nucleic acid acting as a universal internal control. A primer pair includes a first primer having SEQ ID NO: 1 or a complement thereof and a second primer having SEQ ID NO: 2 or a complement thereof. A probe includes SEQ ID NO: 3 or a complement thereof, preferably wherein each of the nucleotides in position 4, 5, 6, 8, 9, 10, 11 and 12 of SEQ ID NO: 3 is replaced with a corresponding locked nucleic acid (LNA) unit (SEQ ID NO: 4).