Nucleic Acid Substrate Protection for Microbial Detection
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Solution Overview
Problem
Current methods for detecting microorganisms in samples, particularly in blood cultures, face challenges such as contamination from host cells and dead microorganisms, low sensitivity in detecting yeast, and the need for prolonged incubation times, leading to unnecessary antibiotic therapy and increased antimicrobial resistance.
Innovation Solution
A method involving pre-modified nucleic acid molecules protected from nuclease activity, increased nucleic acid substrate concentration, and controlled high pH treatment to differentiate microbial from non-microbial nucleic acid modifying activities, allowing for rapid and accurate detection of microorganisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If culture approaches are used to detect small numbers of bacteria, then detection capability is improved, but incubation time increases to several days
Solution Approach 1:
The patent applies preliminary action by performing nucleic acid extraction and amplification before bacterial growth is visible on culture media. The method extracts nucleic acids from the sample, amplifies them through PCR, and detects them before the bacteria would normally be detectable by culture methods, thereby reducing incubation time while maintaining detection capability for small numbers of bacteria.
Solution Approach 2:
The patent replaces the mechanical/cultural system of growing bacteria on media with a molecular biology system using nucleic acid amplification. Instead of relying on bacterial growth and visual inspection, the method uses PCR to amplify and detect nucleic acids, enabling rapid detection without the several-day incubation required for culture approaches.
2Productivity
If ETGA assays are used to detect viable microorganisms, then detection speed is improved, but false positives increase due to contaminating polymerase activity from host cells and dead microorganisms
Solution Approach 1:
The patent applies local quality by using primers with specific sequence characteristics that are adapted to bind selectively to microbial nucleic acids. The primers are designed with sequences that have optimal binding affinity for microbial DNA/RNA while having reduced affinity for host cell nucleic acids, thereby achieving specific detection that minimizes false positives from contaminating polymerase activity.
Solution Approach 2:
The patent uses nucleic acid amplification as an intermediary step between sample processing and detection. The amplified nucleic acid sequences serve as intermediaries that can be detected with high sensitivity and specificity, allowing the system to distinguish between viable microorganisms and contaminants through the amplification process itself.
3Reliability
If high pH treatment is used to remove contaminating enzyme activity, then specificity is improved, but sensitivity decreases for certain bacterial strains such as H. influenzae
Solution Approach 1:
The patent replaces the chemical high pH treatment method with a molecular biology-based specificity approach using designed primers and nucleic acid amplification. Instead of relying on pH conditions that can harm certain bacterial strains, the method uses sequence-specific priming and amplification to achieve specificity, thereby maintaining sensitivity for all bacterial strains including H. influenzae.
4Productivity
If rapid testing methods are implemented, then productivity is improved, but the ability to determine absence of microorganisms is compromised
Solution Approach 1:
The patent applies continuity of useful action by implementing a rapid nucleic acid amplification method that can determine both presence and absence of microorganisms through a continuous, streamlined process. The method maintains high productivity while reliably determining absence by using sensitive detection that can identify even low levels of nucleic acid, allowing rapid differentiation between positive and negative samples without compromising the ability to confirm absence.
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 enhances sensitivity and specificity, reduces false positives, and enables faster determination of microorganism absence or presence, thereby reducing unnecessary antibiotic therapy and antimicrobial resistance.
Implementation Method 1
the nucleic acid molecule is modified so as to protect it from nuclease activity
Implementation Method 2
contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample
Implementation Method 3
treating the sample under high pH conditions for no more than 5 minutes in order to inhibit the non-microbial source of nucleic acid modifying activity
Data Source
AI summary
Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism.Corresponding kits are also provided.