EV-D68 Detection Using VP1-Targeted Primers and Probes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting enterovirus D68 (EV-D68) are inadequate in specificity and sensitivity, particularly in distinguishing EV-D68 from other enteroviruses, which can lead to false positives and negatives during outbreaks, such as the 2014 outbreak in the United States.
Innovation Solution
The use of specific primers and probes designed to target the viral protein 1 (VP1) nucleic acid of EV-D68, allowing for real-time PCR or rRT-PCR detection, ensuring high specificity and sensitivity by amplifying and hybridizing only to EV-D68 sequences, thereby accurately identifying the virus in samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for enterovirus, then detection can be performed, but specificity is insufficient leading to false positives and inability to distinguish EV-D68 from other enteroviruses
Solution Approach 1:
The patent applies local quality by designing primers and probes that target specific local regions of the EV-D68 VP1 nucleic acid sequence. The forward primer targets nucleotides 2518-2542, the reverse primer targets nucleotides 2761-2789, and the probe targets nucleotides 2647-2669 of the EV-D68 VP1 gene. This localized targeting of specific sequence regions enables high specificity detection of EV-D68 while distinguishing it from other enterovirus types.
2Measurement precision
If detection methods lack sensitivity, then detection can be performed, but sensitivity is insufficient leading to false negatives during outbreaks
Solution Approach 1:
The patent replaces conventional detection mechanisms with real-time PCR and rRT-PCR amplification systems. These molecular biology techniques use thermal cycling and enzymatic amplification to exponentially increase the target nucleic acid sequences, enabling detection of even minute amounts of EV-D68 genetic material with high sensitivity and reliability, thereby minimizing false negatives during outbreaks.
3Productivity
If non-specific primers and probes are used, then detection can be performed, but false positives occur due to cross-reactivity with other enteroviruses
Solution Approach 1:
The patent segments the detection system into three distinct functional components: a forward primer (nucleotides 2518-2542), a reverse primer (nucleotides 2761-2789), and a fluorescent probe (nucleotides 2647-2669). Each component is designed to recognize specific segments of the EV-D68 VP1 nucleic acid sequence. This segmentation allows for highly specific detection where all three components must bind to their respective target sequences, dramatically reducing false positives from cross-reactivity with other enteroviruses.
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
The described methods and reagents enable precise detection of EV-D68, including the 2014 North America lineage, with high sensitivity and specificity, reducing false positives and negatives, and effectively identifying the virus in clinical and laboratory settings.
Implementation Method 1
contacting a sample with at least one primer (such as a forward primer and/or a reverse primer) capable of specifically amplifying an EV-D68 viral protein 1 (VP1) nucleic acid
Implementation Method 2
a detectably labeled probe capable of specifically hybridizing to an EV-D68 VP1 nucleic acid under conditions sufficient for specific amplification of the EV-D68 VP1 nucleic acid by the at least one primer and/or under conditions sufficient for specific hybridization of the probe to the EV-D68 nucleic acid
Data Source
AI summary
Methods and compositions for detection of enterovirus D in a sample, particularly detection of enterovirus D68, are provided. The methods include contacting a sample with at least one primer (such as a forward primer and/or a reverse primer) capable of specifically amplifying an EV-D68 viral protein 1 (VP1) nucleic acid or a portion thereof and/or a detectably labeled probe capable of specifically hybridizing to an EV-D68 VP1 nucleic acid, under conditions sufficient for specific amplification of the EV-D68 VP1 nucleic acid by the at least one primer and/or under conditions sufficient for specific hybridization of the probe to the EV-D68 nucleic acid. The amplification of the EV-D68 VP1 nucleic acid and/or the hybridization of the probe to the EV-D68 VP1 nucleic acid is detected, thereby identifying presence of EV-D68 in the sample.