Enterovirus Detection Using 5'UTR Targeted Primers
Solution Overview
Problem
Current diagnostic methods for enterovirus infections, such as those caused by rhinoviruses, face challenges due to high sequence similarity, leading to misidentification and difficulties in distinguishing between different viral species, especially in cases of co-infections, which complicates both clinical diagnosis and epidemiological studies.
Innovation Solution
The use of specific PCR primer pairs and sequencing primers targeting the 5' untranslated region (5'UTR) of enteroviruses allows for the identification, subtyping, and classification of viral infections in a cost-effective and high-throughput manner, utilizing nucleic acid sequencing and pyrosequencing technology to provide reliable data for microbial genotyping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If standard qPCR methods are used for enterovirus detection, then detection speed is improved, but measurement precision deteriorates due to cross-priming and misidentification
Solution Approach 1:
The patent segments the detection process into two distinct stages: (1) a screening stage using standard qPCR with universal primers for rapid detection, and (2) a confirmation stage using sequencing with type-specific primers for accurate identification. This segmentation allows the system to benefit from both the speed of qPCR and the precision of sequencing-based methods.
Solution Approach 2:
The patent introduces an intermediary step between qPCR detection and final identification: the use of type-specific primers for sequencing. These primers act as a mediator that bridges the gap between rapid detection and precise identification, allowing the system to resolve cross-priming issues while maintaining workflow efficiency.
2Ease of manufacture
If standard qPCR methods are used for enterovirus detection, then cost effectiveness is improved, but reliability deteriorates due to inability to detect co-infections
Solution Approach 1:
The patent applies partial action by using sequencing only for samples that test positive in the initial qPCR screen. This selective application of the more expensive and time-consuming sequencing method to only necessary cases maintains cost-effectiveness while improving reliability for detecting co-infections and accurate virus typing.
3Measurement precision
If nucleic acid sequencing with type-specific primers is used, then measurement precision is improved, but productivity deteriorates due to lower throughput
Solution Approach 1:
The patent uses sequencing with type-specific primers only for samples that require further characterization after positive qPCR screening, rather than applying it to all samples. This partial application maintains high measurement precision for subtyping while preserving overall productivity by avoiding unnecessary sequencing of negative or clearly identified cases.
Solution Approach 2:
The patent segments the patient population into different groups based on qPCR results: (1) negative samples requiring no further action, (2) positive samples with clear identification, and (3) positive samples requiring subtyping. This segmentation allows the high-precision sequencing method to be applied only to the necessary subset of cases, maintaining both accuracy and throughput.
4Ease of operation
If standard methods are used for viral genotyping, then ease of operation is improved, but measurement precision deteriorates due to unknown mutations affecting primer hybridization
Solution Approach 1:
The patent introduces type-specific primers as an intermediary that overcomes the limitations of universal primers. These primers are designed to target conserved regions while accommodating known variations, serving as a bridge between the simplicity of PCR-based methods and the accuracy needed for precise genotyping despite mutations.
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 enables accurate and efficient identification of enteroviruses, including rhinoviruses, in clinical samples, reducing misidentification and improving the ability to detect co-infections, thereby enhancing diagnostic precision and molecular epidemiological assessments.
Implementation Method 1
The use of specific PCR primer pairs and sequencing primers targeting the 5' untranslated region (5'UTR) of enteroviruses allows for the identification, subtyping, and classification of viral infections
Implementation Method 2
utilizing nucleic acid sequencing and pyrosequencing technology to provide reliable data for microbial genotyping
Implementation Method 3
utilizing nucleic acid sequencing and pyrosequencing technology to provide reliable data for microbial genotyping
Data Source
AI summary
Certain embodiments of the invention include, but are not limited to PCR primer pairs, sequencing primers, and/or associated thermocycling protocols targeting a region identified by the inventors within the 5′ untranslated region of enteroviruses for the purpose of identifying, subtyping, and/or classifying of virus in samples using nucleic acid sequencing. The sequencing procedures can use nucleic acid templates, such as cDNA or PCR amplicons, as a template for sequencing in medium to high throughput format that is cost effective and easily deployed to other clinical microbiology laboratories.