Liquid Chromatography Microbial RNA Isolation via Void Volume
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Solution Overview
Problem
Current methods for detecting microbial species in biological specimens are time-consuming and inefficient, requiring incubation and purification steps that can lead to degradation of genetic biomarkers and inability to distinguish between live and dead organisms, especially in complex clinical and environmental samples.
Innovation Solution
A method utilizing liquid chromatography for bulk filtration of microbial RNA from biological specimens, focusing on the void volume to isolate and collect microbial RNA without intentional growth or purification, allowing for rapid detection and identification of pathogenic organisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If incubation and purification techniques are used to detect microbial species, then detection accuracy is improved, but detection time increases significantly (up to 30 hours)
Solution Approach 1:
The patent applies preliminary action by performing RNA extraction and library preparation before sequencing. The method prepares the biological specimen by extracting total RNA and constructing a sequencing library in advance, allowing the actual detection to be performed rapidly without requiring time-consuming incubation steps. This preliminary preparation enables the detection to be completed in significantly less time while maintaining accuracy.
Solution Approach 2:
The patent applies extraction by isolating and separating microbial RNA from the complex biological specimen matrix. The method extracts total RNA from the specimen and uses rRNA depletion to remove host RNA, enriching for microbial RNA. This extraction process separates the target microbial RNA from interfering substances, enabling accurate and rapid detection without requiring lengthy purification and incubation steps.
2Measurement precision
If purification techniques are used to isolate microbial RNA, then detection precision is improved, but RNA degradation occurs
Solution Approach 1:
The patent replaces mechanical purification techniques with a biochemical approach using rRNA depletion. Instead of using column-based purification or precipitation methods that can cause RNA degradation, the method uses enzymatic or chemical depletion of rRNA to enrich microbial RNA. This substitution maintains RNA integrity while achieving the necessary purification for accurate detection.
Solution Approach 2:
The patent introduces rRNA depletion as an intermediary step between RNA extraction and sequencing. This intermediary process selectively removes host rRNA while preserving microbial RNA, serving as a bridge that enables accurate detection without requiring harsh purification conditions that could degrade the RNA. The rRNA depletion acts as a gentle selective filter that maintains RNA integrity.
3Adaptability or versatility
If conventional detection methods are used on complex biological specimens, then comprehensive analysis is achieved, but ability to distinguish live vs dead organisms is lost
Solution Approach 1:
The patent applies local quality by focusing the analysis on specific RNA characteristics that indicate organism viability. The method examines the quality and integrity of extracted RNA, using metrics such as RNA degradation patterns and presence of specific markers to distinguish between live and dead organisms. This localized focus on quality indicators enables viability assessment while maintaining comprehensive analysis of the microbial community.
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 significantly reduces the time and effort required for microbial RNA detection, effectively amplifying microbial RNA relative to host RNA, enabling timely and accurate identification of live microbial species without degrading biomarkers.
Implementation Method 1
using liquid chromatography to bulk filter microbial RNA molecules from a mixture of RNA molecules in the test sample
Data Source
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AI summary
Detection and identification of microbial species uses liquid chromatography as a bulk filtration process for the rapid isolation and collection of microbial RNA from a biological specimen. In various embodiments, the gene sequencing of microbial RNA molecules from a biological specimen is enhanced by obtaining and then preparing the biological specimen as a test sample for liquid chromatography that is used to bulk filter microbial RNA molecules from a mixture of RNA molecules in the test sample to isolate and collect the microbial RNA molecules in two or more fraction outputs, wherein at least one of the two or more fraction outputs is a fraction within a void volume of the liquid chromatography, preparing one or more fraction outputs for gene sequencing, including the fraction output that is within the void volume, and conducting gene sequencing on the one or more prepared outputs to detect microbial RNA from the biological specimen.