Immunochromatographic Strip Nucleic Acid Detection Internal Control
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Solution Overview
Problem
The presence of large amounts of target nucleic acid in a sample can interfere with the successful co-amplification of an internal control sequence, leading to reduced amplification and misinterpretation of results in nucleic acid amplification tests, potentially resulting in false negatives.
Innovation Solution
The method involves performing nucleic acid amplification reactions with split probes and dual capture zones to ensure the detection of both target and internal control nucleic acids, even in the presence of excess target nucleic acid, by using distinct labels and capture zones to enhance the visibility of the internal control line.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If target nucleic acid is present in large amounts in the sample, then the sensitivity and detection capability of the assay is improved, but the amplification of internal control sequence is inhibited leading to false negatives
Solution Approach 1:
The assay is divided into separate functional components: target-specific probes and internal control probes with distinct labeling schemes. The internal control uses a different fluorophore or labeling system than the target probe, allowing independent detection and visualization of control amplification separate from target amplification.
Solution Approach 2:
An internal control sequence is introduced as an intermediary element that competes with the target sequence for amplification resources. This control sequence is designed to be amplifiable under the same conditions but detectable through a different signaling mechanism, serving as a mediator to monitor amplification success independently of target abundance.
2Reliability
If internal control sequence is co-amplified with target nucleic acid, then the reliability of results is improved by detecting inhibition, but the amplification efficiency of both sequences is reduced due to competitive interference
Solution Approach 1:
The concentrations of primers, probes, and nucleic acid templates are optimized to achieve balanced amplification of both target and internal control sequences. The internal control is designed with specific sequence characteristics and concentration levels that allow it to amplify efficiently even in the presence of excess target sequence, maintaining detectable signal levels for both.
Solution Approach 2:
The internal control sequence is present in excess relative to typical target concentrations, ensuring that even when target sequence dominates the reaction, sufficient internal control amplicon is generated to produce a detectable signal. This excessive presence of control template compensates for the competitive disadvantage.
3Device complexity
If a single probe system is used for detection, then the device complexity is reduced, but the ability to distinguish target from internal control signals is lost
Solution Approach 1:
Different fluorophores or labeled probes are used to detect target and internal control sequences, producing distinguishable color or fluorescence signals. For example, the target probe may be labeled with FITC (green fluorescence) while the internal control probe is labeled with a different fluorophore (red fluorescence), allowing simultaneous detection and clear differentiation of both signals using standard fluorescence detection equipment.
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 ensures the successful amplification and detection of both target and internal control nucleic acids, preventing false negatives and allowing for accurate interpretation of results, even when the internal control amplification is reduced due to competitive interference from excess target nucleic acid.
Implementation Method 1
nucleic acid probes useful in combination to detect target and control nucleic acids. A first nucleic acid probe includes a ligand and a sequence complementary to a target nucleic acid. A second nucleic acid probe includes the same ligand and a sequence complementary to a different, control nucleic acid.
Implementation Method 2
The hybridized probe can be visualized through a variety of methods well known in the art that include the labeling of the probe with enzymes or luminescent or fluorescent reagents. The detection of an amplified product with luminescent or fluorescently labeled probes requires the use of specialized equipment
Data Source
AI summary
Compositions and methods useful in nucleic acid assays are provided. The invention permits detection of test and control nucleic acids. Test nucleic acids can be immobilized at multiple locations, such that amplification of either a test nucleic acid or a control nucleic acid provides a captured nucleic acid in a control capture zone.