Signal Suppressor Nucleic Acid for False Positive Elimination
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Solution Overview
Problem
Current methods for detecting bacterial species in clinical samples face challenges in distinguishing between viable, disease-causing pathogens and non-viable or contaminating pathogens, particularly in end point detection approaches, due to issues with precision, sensitivity, and the ability to quantify viable organisms.
Innovation Solution
A kit and method utilizing a signal suppressor nucleic acid, DNA primers, and a nucleic acid amplification process to determine if a target cell population is present above a threshold level, preventing false positives from contaminants by ensuring amplification only occurs if the target is above a certain quantity, using helicase-dependent amplification or PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR cycle threshold method is used to detect target nucleic acid, then detection sensitivity is improved, but imprecision in Ct value occurs due to sample components impacting amplification efficiency
Solution Approach 1:
A signal suppressor nucleic acid is introduced as an intermediary substance that competes with target nucleic acid for primer binding. This suppressor is added in controlled amounts to consume excess primers, thereby suppressing amplification of low-level targets below a predetermined threshold while allowing amplification of significant targets to proceed, thus resolving the precision issue caused by variable sample components
Solution Approach 2:
The invention changes the parameter of primer availability by controlling the ratio of primers to signal suppressor. By adjusting the amount of signal suppressor added, the system modifies the effective primer concentration available for target amplification, creating a threshold effect that improves both sensitivity and precision simultaneously
2Reliability
If propidium monoazide crosslinking method is used to distinguish viable organisms, then specificity is improved by preventing amplification of dead cell DNA, but sensitivity decreases because only cells with compromised membranes have their DNA exposed
Solution Approach 1:
The signal suppressor acts as an intermediary that selectively suppresses amplification based on quantity rather than viability state. By competing for primers in proportion to target abundance, it enables detection of viable organisms without requiring membrane compromise, thus maintaining sensitivity while achieving specificity through threshold-based discrimination
Solution Approach 2:
The invention segments the detection range by creating a threshold boundary. Targets above the threshold (significant quantities) are detected while targets below the threshold (contaminants or dead cells) are suppressed, achieving both sensitivity and specificity without relying on membrane integrity
3Device complexity
If end point detection is used to amplify nucleic acid, then device complexity is reduced compared to real-time detection, but measurement precision deteriorates due to inability to determine when to stop amplification
Solution Approach 1:
The signal suppressor is added preliminarily to the reaction mixture before amplification begins. This pre-established competitive inhibition creates a built-in threshold mechanism that allows end-point detection systems to accurately distinguish significant targets from contaminants without requiring complex real-time monitoring equipment
Solution Approach 2:
The amplification reaction itself provides the detection signal at the endpoint. The signal suppressor enables the reaction to self-discriminate between significant and insignificant targets based on whether amplification overcomes the suppressor competition, eliminating the need for external quantitation controls
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 effectively differentiates between viable and non-viable pathogens, reducing false positives and improving detection sensitivity by ensuring that only significant quantities of target nucleic acid are amplified, thus accurately determining the presence of disease-causing organisms.
Implementation Method 1
subjecting the sample to a nucleic acid amplification process, cause the upstream and downstream DNA primers to amplify a sequence between the upstream and downstream annealing sites of the nucleic acids in both the signal suppressor and the target cell population
Implementation Method 2
using helicase-dependent amplification or PCR
Implementation Method 3
using helicase-dependent amplification or PCR
Implementation Method 4
the signal suppressor is provided in a sufficient quantity to eliminate detectable levels of amplification of target cell nucleic acid if the target cell population is present below a threshold amount
Data Source
AI summary
Methods, materials, and kits for distinguishing a population of cells or organisms truly present in a clinical specimen from contaminating cells or organisms is disclosed. The methods and kits use a suppressor to avoid false positive detection of contaminants in nucleic acid amplification reactions.