RT-LAMP Primer Sets for Rapid SARS-CoV-2 Gene Detection
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Solution Overview
Problem
Current methods for diagnosing SARS-CoV-2, such as RT-PCR, are time-consuming and lack sensitivity and specificity, necessitating the development of faster and more sensitive diagnostic tools for use in laboratory-independent healthcare settings.
Innovation Solution
A set of primers for RT-LAMP (reverse transcription loop-mediated isothermal amplification) targeting the N and S genes of SARS-CoV-2, combined with fluorescent dyes, enabling rapid and sensitive detection of the virus with a limit of 50 genome equivalents per microliter in less than 20 minutes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RT-PCR method is used for SARS-CoV-2 diagnosis, then detection sensitivity is improved, but diagnostic time increases
Solution Approach 1:
The patent changes the fundamental parameter of the amplification process from thermal cycling (PCR) to isothermal amplification (LAMP). By maintaining a constant temperature of 65°C instead of cycling through multiple temperature phases, the method achieves both rapid amplification (within 20 minutes) and high detection sensitivity (50 genome equivalents per microliter), resolving the contradiction between speed and sensitivity
Solution Approach 2:
The patent extracts and utilizes specific conserved regions from both the N gene (nucleocapsid) and S gene (spike) of SARS-CoV-2 to design targeted primers. By focusing amplification on these specific genomic regions rather than the entire genome, the method achieves rapid and sensitive detection without requiring the time-consuming full genome analysis performed by traditional RT-PCR
2Measurement precision
If RT-PCR method is used for SARS-CoV-2 diagnosis, then detection specificity is improved, but diagnostic time increases
Solution Approach 1:
The patent designs a multi-functional diagnostic system that can detect multiple SARS-CoV-2 variants simultaneously by targeting conserved regions in both N and S genes. The isothermal LAMP methodology itself serves multiple functions: amplification, detection, and differentiation of viral variants, all within a single 20-minute reaction without requiring complex thermal cycling equipment
Solution Approach 2:
The patent changes the temperature parameter from variable (cycling) to constant (isothermal at 65°C), which simplifies the diagnostic process while maintaining high specificity through carefully designed primers that target conserved viral sequences, enabling rapid and accurate diagnosis in under 20 minutes
3Measurement precision
If complex diagnostic equipment is used, then detection accuracy is improved, but ease of operation decreases
Solution Approach 1:
The patent employs a self-service diagnostic approach where the LAMP reaction mixture contains all necessary components (primers, nucleotides, buffer, and detection reagents) in a single tube. The reaction is automatically initiated by adding the viral sample and proceeds isothermally without requiring complex temperature control, making the system easy to operate while maintaining high detection accuracy through the inherent specificity of the primer design
Solution Approach 2:
The patent merges multiple diagnostic functions into a single integrated LAMP reaction system. The same reaction tube performs viral RNA amplification, fluorescence detection, and result interpretation, eliminating the need for separate equipment for each function and greatly simplifying operation while preserving detection accuracy through the robust isothermal amplification chemistry
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 primers provide highly sensitive and specific detection of SARS-CoV-2, allowing for rapid diagnosis in laboratory-independent settings using portable genetic analyzers, with the ability to quantify viral RNA levels.
Implementation Method 1
reverse transcription loop-mediated isothermal amplification (RT-LAMP) method
Implementation Method 2
Loop-mediated isothermal amplification (LAMP) method is a single-tube technique for the amplification of DNA/RNA
Implementation Method 3
combined with fluorescent dyes, enabling rapid and sensitive detection of the virus
Data Source
Figure 1~2
Figure 3(1)~4
Figure 5~6
AI summary
Provided is a primer set for detecting the N gene of SARS-CoV-2 using a reverse transcription loop- mediated isothermal amplification (RT-LAMP) method, comprising at least a first F3 primer comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 1, at least a first B3 primer comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 2, at least a first F2 primer comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 3, at least a first B2 primer comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 4, at least a first F1c primer comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 5 or to SEQ ID NO: 8, at least a first B1c primer comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 6. Also provided are kits and methods for using the described primers, and a primer set for similarly detecting the S gene of SARS-CoV-2.