Colorimetric Saliva SARS-CoV-2 Detection via LAMP Isothermal Amplification
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Solution Overview
Problem
Current COVID-19 testing methods are inadequate for rapid, on-site detection of SARS-CoV-2 infections in saliva samples, as they require RNA extraction and laboratory equipment, leading to delayed results and hindered throughput, especially in identifying asymptomatic carriers.
Innovation Solution
A method using loop-mediated isothermal amplification (LAMP) primers to detect SARS-CoV-2 RNA in saliva samples, involving dilution with a base, mixing with a lysis buffer containing protease and denaturing agents, and analyzing the color change of the test mixture, which can be performed without RNA extraction or laboratory equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RT-qPCR testing is used, then detection accuracy is improved, but testing time and device complexity increase
Solution Approach 1:
The patent extracts the essential detection function from complex laboratory equipment by using a simplified isothermal amplification system that operates without thermal cyclers. The method isolates the core amplification and detection steps, removing the need for RNA extraction and complex laboratory equipment, thereby reducing testing time while maintaining detection accuracy for SARS-CoV-2 RNA in saliva samples.
Solution Approach 2:
The patent changes the temperature parameter from cyclic heating and cooling in RT-qPCR to a constant isothermal temperature (65°C) for the LAMP reaction. This parameter change simplifies the thermal regime, eliminates the need for thermal cyclers, and reduces testing time while maintaining detection sensitivity through the colorimetric pH indicator system.
2Measurement precision
If conventional RT-qPCR testing is used, then detection accuracy is improved, but device complexity increases
Solution Approach 1:
The patent extracts the essential detection function from complex laboratory equipment by using a simplified isothermal amplification system that operates without thermal cyclers. The method isolates the core amplification and detection steps, removing the need for RNA extraction and complex laboratory equipment, thereby reducing testing time while maintaining detection accuracy for SARS-CoV-2 RNA in saliva samples.
Solution Approach 2:
The patent replaces the mechanical thermal cycling system with a simple constant temperature water bath or heating block. The complex temperature cycling mechanism is substituted with a straightforward isothermal heating approach, and the fluorescence detection system is replaced with a simple colorimetric readout using pH indicators, dramatically reducing device complexity.
3Productivity
If rapid testing is implemented, then testing throughput is improved, but detection sensitivity may worsen
Solution Approach 1:
The patent optimizes the primer concentration, Mg2+ concentration, and reaction time parameters to achieve rapid amplification within 15-30 minutes while maintaining high detection sensitivity. The colorimetric pH indicator system is tuned to detect even subtle pH changes caused by limited amplification, ensuring that rapid testing does not compromise sensitivity for detecting as low as 2.5 copies of SARS-CoV-2 RNA/μL.
Solution Approach 2:
The patent employs a composite reaction system combining LAMP primers, Betalactamase enzyme, and pH indicators (phenol red and/or bromothymol blue) that work synergistically. The Betalactamase enzyme provides continuous amplification during the reaction, enhancing sensitivity, while the pH indicators provide visual detection. This composite approach enables rapid testing with high sensitivity and throughput.
4Ease of operation
If saliva samples are used, then ease of sample collection is improved, but sample quality for RNA extraction worsens
Solution Approach 1:
The patent extracts and eliminates the RNA extraction step entirely by using a direct LAMP amplification approach on saliva samples. The method processes saliva directly with LAMP reagents after simple dilution and neutralization, removing the need for complex RNA extraction procedures while maintaining detection reliability for SARS-CoV-2 RNA.
Solution Approach 2:
The patent adjusts the pH parameter by adding base (NaOH or KOH) to neutralize acidic saliva samples, creating optimal conditions for LAMP amplification directly in saliva. This parameter adjustment ensures reliable detection despite the variable composition of saliva, maintaining sample quality without requiring extraction.
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 method enables rapid, point-of-care COVID-19 testing with high sensitivity and specificity, capable of detecting as low as 2.5 copies of SARS-CoV-2 RNA/μL, reducing turn-around times and increasing throughput, while avoiding cross-reactivity with other pathogens.
Implementation Method 1
contacting the diluted saliva sample with loop-mediated isothermal amplification (LAMP) primers that bind the N gene of the SARS-CoV-2 virus to form a test mixture under conditions that allow for reverse-transcription LAMP (RT-LAMP) of RNA material within the diluted saliva sample to take place
Implementation Method 2
analyzing the color of the test mixture
Data Source
AI summary
The present disclosure provides a method of testing a subject for COVID-19, the method comprising the steps of: obtaining a saliva sample from the subject; diluting the saliva sample with a dilution buffer comprising a base; contacting the diluted saliva sample with one or more loop-mediated isothermal amplification (LAMP) primers that bind regions in the N gene of the SARS-CoV-2 virus to form a test mixture; and analyzing the color of the test mixture. The present disclosure also provides a COVID-19 rapid-test kit comprising one or more components for the collection of a saliva sample from a subject, one or more components for processing of the saliva sample, and one or more components used to interpret the results of the COVID-19 test.