Nucleic Acid Internal Control Reagents for NAT Assay Reliability
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Solution Overview
Problem
Existing nucleic acid testing (NAT) assays face challenges in identifying robust internal control sequences, primers, and probes that are sensitive, reproducible, and versatile enough to detect and quantify a wide array of target sequences, due to issues like hairpins, A/T runs, and narrow reaction condition ammenability.
Innovation Solution
The development of internal control reagents, including polynucleotides, oligonucleotides, and kits that provide highly sensitive and reproducible nucleic acid sequences, primers, and probes, such as those with specific sequences (SEQ ID NOs: 1, 8, 9, 10, 11, 12, 13, 14, 15) and variants, for use in nucleic acid amplification reactions, including quantitative PCR, to serve as templates, primers, and probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional internal control nucleic acid sequences are used, then the assay can detect target nucleic acids, but false positives or false negatives occur due to hairpins, A/T runs, or G/C runs affecting amplification and probe hybridization
Solution Approach 1:
The patent applies parameter changes by modifying the nucleic acid sequence composition to achieve optimal GC content (40-60%) and avoid problematic regions. The internal control sequences are specifically designed with controlled nucleotide composition to prevent hairpin formation and ensure proper annealing temperatures, thereby eliminating false positives/negatives while maintaining detection accuracy
Solution Approach 2:
The patent creates simplified copy versions of internal control sequences that replicate the essential amplification and hybridization functions without the harmful structural features. These copied sequences are optimized to lack hairpins and extreme A/T or G/C runs, providing reliable control signals without the artifacts of conventional sequences
2Measurement precision
If a specific internal control template sequence is designed for high sensitivity, then detection of low copy numbers is achieved, but the control becomes incompatible with multiplex reactions or requires extensive empirical validation
Solution Approach 1:
The patent achieves universality by designing internal control sequences that function reliably across diverse reaction conditions and multiplex assays. The sequences are engineered with balanced GC content and optimal lengths to maintain consistent amplification efficiency whether used alone or with multiple target sequences, eliminating the need for separate validation for each application
Solution Approach 2:
The patent optimizes sequence parameters including length (100-1000 nucleotides), GC content (40-60%), and nucleotide distribution to achieve broad compatibility. These parameter adjustments ensure the internal control maintains high detection sensitivity while adapting to various thermal cycling conditions and multiplex reaction environments without requiring extensive re-validation
3Productivity
If internal control sequences are optimized for narrow reaction conditions, then amplification efficiency is high, but the control cannot be used in a variety of multiplex reactions
Solution Approach 1:
The patent balances amplification efficiency with versatility by optimizing key parameters: GC content set to 40-60% for stable yet adaptable hybridization, sequence length adjusted to 100-1000 nucleotides for efficient amplification across conditions, and nucleotide distribution engineered to avoid extreme temperature requirements. This parameter optimization enables the internal control to maintain high productivity across broad reaction condition ranges
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
These internal control reagents exhibit high sensitivity and reproducibility, enabling accurate detection and quantification of target sequences across various conditions, improving the reliability of NAT assays and allowing for multiplex reactions.
Implementation Method 1
polynucleotides that can be used as templates in nucleic acid amplification reactions
Implementation Method 2
quantitative PCR
Implementation Method 3
contacting the template sequence with at least one primer that specifically hybridizes to the template sequence
Implementation Method 4
extending the primer, thus producing an amplicon of the template sequence
Implementation Method 5
contacting the amplicon with a probe, wherein the probe binds to the amplicon
Implementation Method 6
The probe comprises a fluorophore and a quencher
Data Source
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AI summary
The present invention relates to nucleic acids sequences that can be used for nucleic acid amplification, for example quantitative nucleic acid amplification.