Discriminating Positive Control Nucleic Acid for Microbial Detection
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Solution Overview
Problem
Current methods for detecting microorganisms in samples, such as food and environmental samples, face challenges in distinguishing true positives from false positives and in efficiently detecting low copy numbers of microorganisms due to contamination and limitations in nucleic acid amplification techniques.
Innovation Solution
A method involving the use of a discriminating positive control nucleic acid sequence that is mixed with the test sample, extracted, and amplified in the presence of an intercalating dye, allowing for differentiation through melt curve analysis to confirm the presence or absence of microorganisms by generating distinct melt curves for the control and microorganism amplicons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If actual pathogen nucleic acid controls are used in detection assays, then the sensitivity and ability to detect true positives is improved, but the risk of contamination of test samples and false positive results increases
Solution Approach 1:
The patent divides the control function into separate components: a positive control nucleic acid sequence that is distinct from the pathogen target sequence. This segmentation allows the control to be amplified and detected separately, preventing contamination of test samples while maintaining the ability to detect true positives through distinct melt curve analysis.
Solution Approach 2:
The patent introduces an intermediary control nucleic acid sequence that serves as a mediator between the amplification reaction and the detection system. This control sequence is amplified alongside the pathogen target but produces a distinguishable melt curve, acting as a mediator that confirms extraction and amplification without risking false positive results.
2Reliability
If internal control nucleic acid sequences are added to detection assays, then the confirmation of extraction and amplification is improved, but the complexity of the assay procedure increases
Solution Approach 1:
The patent merges the positive control function with the pathogen detection function by amplifying both the control nucleic acid sequence and the pathogen target sequence in a single PCR reaction. This combining approach confirms extraction and amplification through the control while maintaining assay simplicity, as both amplifications occur simultaneously rather than requiring separate procedures.
Solution Approach 2:
The patent uses melt curve analysis, which can be visualized through temperature-induced changes in the amplification products. The distinct melt curves of the control and pathogen amplicons provide visual differentiation, allowing confirmation of extraction and amplification without adding significant procedural complexity.
3Quantity of substance
If traditional PCR amplification is used for microorganism detection, then the ability to detect microorganisms is improved, but the detection of low copy numbers is compromised due to contamination and false positives
Solution Approach 1:
The patent replaces traditional mechanical separation methods with thermal melt curve analysis. By utilizing temperature-induced denaturation differences between the control and pathogen amplicons, the system achieves precise differentiation even at low copy numbers, eliminating the need for complex mechanical separation procedures.
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 reduces false positive results, confirms nucleic acid extraction, and enables the detection of microorganisms by distinguishing their amplicons from the control amplicons, improving the accuracy and reliability of microbial detection in samples.
Implementation Method 1
amplifying at least some of the extracted nucleic acid and the control nucleic acid wherein amplification is performed in the presence of an intercalating dye
Implementation Method 2
detecting at least some of the amplified nucleic acids, wherein detection comprises measuring a detectable signal of the intercalating dye over a change in temperature; and generating a first melt curve for the discriminatory positive control amplicon and a second melt curve for a microorganism amplicon if present
Data Source
Figure 1
AI summary
The present teachings generally relate to methods and kits incorporating a discriminating positive control for determining whether a particular microorganism or group of microorganisms are present in a sample, for example but not limited to a food, environmental, agricultural, biopharmaceutical, pharmaceutical, or water sample. According to certain methods, at least part of a starting material, for example but not limited to, a food, environmental, agricultural, biopharmaceutical, pharmaceutical, or water sample can be combined with a culture medium and incubated under conditions suitable for microbial growth followed by extracting microorganism and added control nucleic acids for analysis. The extracted nucleic acids are amplified and the amplified nucleic acids are detected, directly or indirectly, and the fidelity of the methods and the presence or absence of the corresponding microorganism is determined because the discriminating positive control provides both confirmatory results for the methods used but eliminates false positive results.