Multiplex rRT-PCR Diagnostic Kit for Coronavirus Detection
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Solution Overview
Problem
Existing diagnostic tools for COVID-19, SARS-CoV, MERS-CoV, and HCoV have limitations such as false negatives/positives, low sensitivity, and long sampling times, posing challenges in accurately diagnosing these viruses, especially with evolving strains like SARS-CoV-2.
Innovation Solution
A multiplex real-time RT-PCR (rRT-PCR) method with specific fluorophores for each target gene, including N1, N2, N3, E, and host cell markers, to enhance sensitivity and specificity, using a diagnostic kit for rapid detection of these viruses in respiratory specimens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional diagnostic tools are used for coronavirus detection, then the diagnostic process can be performed with existing equipment, but the sensitivity is low and false negatives/positives occur frequently
Solution Approach 1:
The patent combines multiple detection targets (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV) into a single multiplex rRT-PCR assay. By merging multiple primer-probe sets targeting different coronavirus genes (N1, N2, N3, E, upE, replicase) into one reaction system with distinct fluorophores, the method achieves high sensitivity and reliability while detecting multiple viruses simultaneously, resolving the contradiction between using existing equipment and achieving high diagnostic accuracy.
Solution Approach 2:
The diagnostic kit is designed with universal applicability to detect multiple coronavirus types and variants through a single platform. The multiplex rRT-PCR method can identify SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV using the same reagent system and equipment, providing both high sensitivity and broad coverage, thus improving reliability without requiring multiple separate tests.
2Measurement precision
If multiple separate diagnostic tests are performed for different coronaviruses, then each virus can be detected with high specificity, but the sampling time becomes excessively long
Solution Approach 1:
The patent merges multiple coronavirus detection assays into a single multiplex rRT-PCR reaction. By combining primer-probe sets for SARS-CoV-2 (N1, N2, N3 targets), SARS-CoV (E gene), MERS-CoV (upE gene), and HCoV (replicase gene) into one test with different fluorophores (FAM, HEX, Texas Red, Cy5), the method maintains high specificity for each virus while reducing diagnostic time from hours to a single rapid test.
3Adaptability or versatility
If a comprehensive diagnostic test for all coronavirus types is developed, then detection coverage is improved, but the device complexity increases
Solution Approach 1:
The diagnostic kit achieves universal detection of multiple coronavirus types through a single multiplex rRT-PCR platform. The system uses a unified buffer system, common enzymatic reagents, and standardized fluorophore detection, allowing broad virus coverage (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV) without proportionally increasing complexity. The method maintains operational simplicity while expanding detection capabilities.
Solution Approach 2:
The patent adds a spectral dimension to the detection system by using multiple fluorophores with distinct emission wavelengths (FAM-green, HEX-yellow, Texas Red-orange, Cy5-red) to differentiate between various coronavirus targets. This dimensional approach allows simultaneous detection of multiple viruses in one reaction without requiring separate physical test systems, thereby increasing versatility while managing complexity through optical differentiation.
4Productivity
If rapid diagnostic testing is implemented, then the detection time is reduced, but the sensitivity and accuracy may be compromised
Solution Approach 1:
The patent employs preliminary optimization of the rRT-PCR protocol including pre-designed primer-probe sets, optimized buffer compositions, and predetermined thermal cycling conditions. The one-step rRT-PCR method integrates reverse transcription and amplification in a single pre-optimized reaction, allowing rapid results within 1-2 hours while maintaining high sensitivity through validated assay conditions and appropriate control mechanisms.
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 method provides rapid, accurate, and specific detection of multiple coronaviruses, reducing false negatives and requiring less than 3 hours for results, suitable for any laboratory with real-time PCR equipment.
Implementation Method 1
each fluorophore having a different emission wavelength... FAM... HEX... Texas Red... and Cy5
Data Source
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AI summary
The invention relates to a diagnostic kit for multiple detection of 4 viruses of the Family Coronaviridae: HCoV, SARS-CoV, MERS-CoV and the SARS-CoV-2 viral strain that has caused a pandemic of the disease known as COVID-19. The kit uses a "One-Step" approach with quantitative gene amplification after backward transcription of the viral genome (rRT-PCR). In order to avoid potential false negatives, the invention contains a double control using Porcine Epidemic Diarrhoea Virus (PEDV -CoV) and Ribonuclease P (RNase P-RP).