Positive Control Nucleic Acid Segmentation for Contamination Detection
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Solution Overview
Problem
Current nucleic acid amplification and detection methods face challenges in preventing contamination of reaction containers with positive control nucleic acids, leading to false-positive results and inaccurate quantification due to the amplification of trace amounts of positive control nucleic acids, especially in high-throughput testing environments.
Innovation Solution
A method using a positive control nucleic acid with a nucleotide sequence that has 70% or lower sequence identity to the target nucleic acid and a probe with a Tm value of 65°C or lower, specifically designed to be amplified using a primer set for the target nucleic acid, allowing for the detection and quantification of the target nucleic acid while confirming the absence of contamination by the positive control nucleic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If positive control nucleic acid is used in nucleic acid amplification and detection, then the reliability of detection results is improved by confirming negative results are not false negatives, but the risk of false-positive results increases due to unintended contamination of reaction containers with positive control nucleic acid
Solution Approach 1:
The positive control nucleic acid sequence is divided into two segments: a primer-binding sequence that is identical to or highly similar to the target nucleic acid (allowing amplification with target-specific primers), and a probe-binding sequence that is completely different from the target nucleic acid (preventing cross-reactivity with target probes). This segmentation allows the positive control to be amplified by target-specific primers while being detectable only by its own specific probe, thus resolving the contradiction between reliability improvement and false-positive prevention.
Solution Approach 2:
Different regions of the positive control nucleic acid are assigned different properties: the 5' region (primer-binding sequence) has high similarity to the target for amplification purposes, while the 3' region (probe-binding sequence) has zero similarity to the target for specific detection purposes. This local differentiation of sequence properties enables the positive control to serve dual functions without causing false positives.
2Productivity
If positive control nucleic acid is handled in high-throughput testing environments with multiple reaction containers in proximity, then the productivity of nucleic acid amplification and detection is improved, but the risk of contamination and false-positive results increases
Solution Approach 1:
The positive control nucleic acid is segmented into primer-binding and probe-binding regions with different sequence properties, allowing it to be amplified in high-throughput environments using target-specific primers while being specifically detected by its own unique probe. This enables productive high-throughput processing while preventing cross-contamination false positives through sequence differentiation.
Solution Approach 2:
A specific probe for the positive control nucleic acid acts as an intermediary that selectively binds only to the positive control's unique sequence region. This intermediary probe mediates between the amplified positive control DNA and the detection system, ensuring that even in high-throughput environments where contamination may occur, only the intended positive control signals are detected, not contaminating target sequences.
3Ease of operation
If positive control nucleic acid is arranged or immobilized in reaction containers for convenience and rapid amplification, then the ease of operation is improved, but the risk of unintended contamination increases leading to false-positive results
Solution Approach 1:
The positive control nucleic acid sequence is segmented such that the primer-binding region resembles the target (enabling convenient amplification with target-specific primers) while the probe-binding region is entirely different (ensuring specific detection). This segmentation allows the positive control to be conveniently pre-immobilized in reaction containers while maintaining safety against false positives through probe-specific detection.
Solution Approach 2:
Different local regions of the positive control nucleic acid have different sequence qualities: the primer-binding region has high sequence identity to the target for amplification convenience, while the probe-binding region has zero sequence identity to the target for detection specificity. This local quality differentiation resolves the contradiction between ease of operation and false-positive prevention in pre-prepared reaction containers.
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 enables convenient and rapid confirmation of container contamination and accurate detection or quantification of target nucleic acids, reducing false-positive results and improving the reliability of nucleic acid amplification and detection tests.
Implementation Method 1
a probe specific for the positive control nucleic acid... wherein the nucleotide sequence of a probe specific for the positive control nucleic acid is a nucleotide sequence that has 70% or lower sequence identity to the sequence of any portion of the genomes of an organism species from which the test sample is derived
Implementation Method 2
performing a nucleic acid amplification reaction using a nucleic acid obtained from a test sample and a primer set specific for a target nucleic acid... performing a nucleic acid amplification reaction using the positive control nucleic acid and the primer set specific for the target nucleic acid
Data Source
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AI summary
An object of the present invention is to provide a set capable of conveniently and rapidly confirming the presence or absence of contamination of a container for a test sample, the set comprising a positive control nucleic acid and a probe specific for the positive control nucleic acid. Another object of the present invention is to provide a method for detecting or quantifying a target nucleic acid in a test sample while conveniently and rapidly confirming the presence or absence of contamination of a container for the test sample with a positive control nucleic acid. The objects are attained by, for example, a set for use in a method for detecting or quantifying a target nucleic acid in a test sample, the set comprising a positive control nucleic acid and a probe specific for the positive control nucleic acid, wherein the nucleotide sequence of the probe is a nucleotide sequence that has 70% or lower sequence identity to the sequence of any portion of the genomes of an organism species from which the test sample is derived and an organism species from which the target nucleic acid is derived, and transcripts thereof, and has a Tm value of 65°C or lower.