Avellino Corneal Dystrophy Allele Detection Substrate
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Solution Overview
Problem
Current methods for detecting Avellino corneal dystrophy are time-consuming and costly, requiring multiple steps and high amounts of reagents, and often miss latent symptoms, leading to vision loss in patients undergoing LASIK surgery due to inadequate genetic diagnosis.
Innovation Solution
A method involving epithelial cells adhered to a substrate, agitated in a lysis solution, followed by incubation and genomic DNA isolation, using specific oligonucleotide primer pairs and probes to detect mutations in the TGFβI gene associated with corneal dystrophy, allowing for same-day detection with reduced reagent use.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time PCR analysis is performed with traditional sample collection and isolation methods, then SNP detection can be achieved, but the process requires longer assay conditions and greater amounts of reagents, resulting in increased costs and reduced productivity
Solution Approach 1:
The patent applies preliminary action by pre-hybridizing oligonucleotide probes to the substrate before sample application. This pre-preparation step allows the actual detection to proceed more rapidly when samples are applied, reducing the overall assay time and increasing productivity while maintaining detection accuracy
Solution Approach 2:
The patent extracts and removes unnecessary steps from the traditional real-time PCR workflow by using a hybridization-based detection method on solid substrates. This eliminates the need for lengthy PCR amplification cycles and complex reagent systems, thereby reducing assay time and reagent consumption while maintaining SNP detection capability
2Measurement precision
If traditional real-time PCR methods are used, then SNP detection is possible, but poor sample quality requires longer assay conditions and greater reagent amounts, increasing costs
Solution Approach 1:
The patent employs disposable solid substrates pre-coated with capture probes that can be discarded after a single use. This eliminates the need for expensive and complex reusable instrumentation and reagent systems required by traditional real-time PCR, significantly reducing reagent consumption and cost while maintaining reliable SNP detection
Solution Approach 2:
The patent replaces the complex mechanical and thermal cycling system of real-time PCR with a simple hybridization-based detection system. This substitution eliminates the need for temperature cycling instruments and complex enzymatic reactions, reducing both equipment costs and reagent consumption while achieving the same diagnostic goal
3Reliability
If real-time PCR assays fail, then additional samples must be collected, causing loss in time and resources
Solution Approach 1:
The patent incorporates feedback mechanisms through the use of internal controls and reference sequences on the substrate that allow immediate assessment of assay validity. This feedback system enables operators to identify successful assays in real-time, eliminating the need for repeat sampling and reducing time losses associated with failed assays
4Measurement precision
If high quality samples are required for real-time PCR, then more stringent collection and isolation procedures are needed, but this increases complexity and reduces ease of operation
Solution Approach 1:
The patent applies partial action by using only the essential components needed for detection - simple buccal swab sampling without complex isolation procedures. The pre-prepared substrate with capture probes compensates for the simplified sample collection, maintaining detection accuracy while dramatically improving ease of operation
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 method enables rapid, precise, and cost-effective detection of Avellino corneal dystrophy alleles, reducing the risk of vision loss by identifying susceptible individuals before LASIK surgery and minimizing the need for additional samples and reagents.
Implementation Method 1
Through the use of differentially labeled fluorescent nucleic acid probes, for example one that binds to a wild type sequence and one that binds to a mutant sequence, single nucleotide changes in the human genome can be quickly and reliably detected
Data Source
AI summary
Systems and methods for detecting at least two genomic alleles associated with corneal dystrophy in a sample from a human subject are disclosed in which cells (e.g., epithelial) of the subject are adhered to a tip of a substrate. The tip of the substrate is agitated in a lysis solution that lyses cells adhered to the substrate. The substrate is removed from the lysis solution upon completion of this agitation. The resulting lysis solution is incubated and then genomic DNA from the lysis solution is isolated to form a gDNA solution. From this, identity of at least two nucleotides present in the human TGFβI gene is determined using at least two oligonucleotide primer pairs and the gDNA solution. These at least two nucleotides are located at respective independent positions of the TGFβI gene corresponding to respective independent single nucleotide polymorphisms (SNPs) associated with corneal dystrophy.