RT-LAMP Primer Sets for Sarbecovirus Detection
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Solution Overview
Problem
Reverse transcriptase qPCR (RT-qPCR) requires significant equipment and time for sample preparation and analysis, making it less accessible for point-of-care diagnostics, especially in resource-limited settings, whereas Loop-Mediated Isothermal Amplification (LAMP) is more simplistic but lacks specific primer sets for reliable detection of Sarbecoviruses like SARS-CoV-2.
Innovation Solution
Development of specific primer sets for reverse transcription loop-mediated isothermal amplification (RT-LAMP) that are at least 85% identical to target nucleotide sequences of Sarbecoviruses, with optimized GC content, end stability, and primer dimerization energy for efficient amplification on solid-reaction media, enabling rapid and accurate detection of Sarbecoviruses like SARS-CoV-2.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RT-qPCR is used for pathogen detection, then detection accuracy is improved, but equipment complexity and sample preparation time increase
Solution Approach 1:
The patent replaces the complex mechanical and optical systems of RT-qPCR (thermal cyclers, fluorescent detection systems) with a simpler isothermal amplification system using LAMP primers that can be performed at constant temperature with visual colorimetric detection, eliminating the need for sophisticated equipment while maintaining diagnostic accuracy
Solution Approach 2:
The patent changes the operational parameters from cyclic temperature changes in RT-qPCR to constant isothermal conditions in LAMP, using specifically designed primers (FIP, BIP, F3, B3, LF, LB) that enable amplification at a single temperature, thereby simplifying the equipment requirements while preserving detection sensitivity
2Measurement precision
If RT-qPCR is used for pathogen detection, then detection accuracy is improved, but time consumption increases
Solution Approach 1:
The patent incorporates reverse transcription and amplification into a single integrated LAMP reaction, performing preliminary RNA-to-cDNA conversion and target amplification simultaneously in one step, thereby eliminating separate preparation stages and reducing total time from sample to result while maintaining diagnostic accuracy
Solution Approach 2:
The patent uses isothermal conditions that allow continuous amplification without repeated heating and cooling cycles, maintaining constant reaction conditions that enable uninterrupted nucleic acid synthesis, thereby reducing the time required to reach detectable amplification levels compared to cyclic PCR methods
3Device complexity
If LAMP is used for pathogen detection, then equipment requirements are reduced, but primer specificity for Sarbecoviruses is insufficient
Solution Approach 1:
The patent designs primers with highly specific local sequence characteristics targeting conserved regions of Sarbecovirus genomes, using optimized GC content, melting temperatures, and absence of secondary structures in specific primer regions to ensure selective binding to Sarbecovirus sequences while preventing cross-reactivity with other viruses
Solution Approach 2:
The patent develops a universal LAMP primer set that can detect multiple Sarbecovirus variants (including SARS-CoV-1, SARS-CoV-2, and related coronaviruses) through a single reaction system, achieving broad-spectrum detection capability while maintaining high specificity through carefully selected target regions that are conserved across the Sarbecovirus genus
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 optimized primer sets enable rapid, reliable, and sensitive detection of Sarbecoviruses, reducing the need for extensive equipment and sample preparation, making RT-LAMP suitable for point-of-care diagnostics with high specificity and low false positives/negatives.
Implementation Method 1
Loop-Mediated Isothermal Amplification (LAMP) is a one-operation nucleic acid amplification method to multiply specific target nucleotide sequences
Implementation Method 2
Reverse-transcriptase LAMP (RT-LAMP) can be used like RT-qPCR in order to identify the presence or absence target nucleotides from RNA
Data Source
AI summary
The present disclosure is drawn to an isolated complementary DNA (cDNA) of a nucleic acid molecule that can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or a combination thereof. In one embodiment, a primer set for reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis can comprise a forward inner primer (FIP) sequence, a backward inner primer (BIP) sequence, a forward outer primer (F3) sequence, a backward outer primer (B3) sequence, a forward loop primer (LF) sequence, and a backward loop primer (LB) sequence. In another embodiment, a method of detecting a target pathogen can comprise providing a primer set.