RT-LAMP CRISPR SARS-CoV-2 Detection System
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Solution Overview
Problem
Current methods for SARS-CoV-2 detection, such as antigen tests and RT-PCR, face challenges in sensitivity, complexity, and cost, particularly in resource-limited settings, necessitating improved methods that are sensitive, specific, and cost-effective without requiring complex instrumentation or highly trained staff.
Innovation Solution
The use of Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) combined with CRISPR-Cas12a chemistry, employing specific nucleic acid sequences and a reporter nucleic acid labeled with fluorophores and quenchers, allows for sensitive and specific detection of SARS-CoV-2 in a single tube format, reducing reagent costs and eliminating the need for thermocyclers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RT-PCR is used for SARS-CoV-2 detection, then sensitivity is improved, but device complexity and cost increase due to requirement of thermocyclers and specialized equipment
Solution Approach 1:
The patent replaces the thermocycler-based thermal cycling system with an isothermal amplification system using Bst polymerase that functions at a constant temperature (65°C). This substitution eliminates the need for complex temperature cycling equipment while maintaining high detection sensitivity through the use of specialized primers and probes designed for isothermal conditions.
Solution Approach 2:
The patent changes the operational temperature parameter from cyclic temperature variations (PCR) to a constant isothermal condition (65°C). This parameter change simplifies the equipment requirements while maintaining amplification efficiency through optimized primer and probe designs that function specifically under isothermal conditions.
2Measurement precision
If RT-PCR is used for SARS-CoV-2 detection, then sensitivity is improved, but cost increases due to specialized equipment and trained staff requirements
Solution Approach 1:
The patent replaces expensive thermocycler equipment with simple water baths or heating blocks that can be obtained through common laboratory channels. This substitution dramatically reduces equipment costs while maintaining detection sensitivity through the use of isothermal amplification chemistry with Bst polymerase and optimized primer-probe sets.
Solution Approach 2:
The patent employs disposable, pre-prepared primer and probe mixes that can be stored at standard refrigerator temperatures. These single-use reagent sets eliminate the need for expensive, durable thermocycler equipment and reduce ongoing operational costs by requiring no specialized maintenance or trained personnel for operation.
3Ease of operation
If antigen tests are used for SARS-CoV-2 detection, then cost and ease of operation are improved, but sensitivity deteriorates for early diagnosis
Solution Approach 1:
The patent employs a self-contained isothermal amplification system that automatically performs RNA extraction, reverse transcription, and amplification in a single reaction tube. The system uses temperature-sensitive reagents that automatically activate at the set temperature, eliminating the need for separate extraction and amplification steps while achieving sensitivity comparable to RT-PCR with ease of use similar to antigen tests.
Solution Approach 2:
The patent merges multiple functions (RNA extraction, reverse transcription, and amplification) into a single isothermal reaction tube. This consolidation maintains the simplicity and point-of-care usability of antigen tests while achieving the high sensitivity of RT-PCR through the combined action of Bst polymerase, reverse transcriptase, and optimized primer-probe sets.
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 approach provides a rapid, sensitive, specific, and cost-effective method for SARS-CoV-2 detection that can be deployed in resource-limited settings, with high diagnostic accuracy and visualizable results, comparable to RT-PCR, while minimizing contamination risks and reagent expenses.
Implementation Method 1
The reporter nucleic acid is preferably a ssDNA or dsDNA labelled at the 5′ and 3′ ends with a fluorophore/quencher pair, the fluorophore being present at one end (5′/3′) and the quencher at the other end (5′/3′)
Data Source
AI summary
Sequences for detection of SARS-CoV-2 are provided and include example, SEQ ID NOs:4-8. The sequences are useful for amplifying the SARS-CoV-2 nsp8 gene in a sample, using Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) and it is preferably used in combination with a Cas enzyme/sgRNA pair and a reporter nucleic acid.The disclosed sequences can be use in methods of detecting SARS-CoV-2 nucleic acids in a sample. Generally, the specific gene sequence of SARS-CoV-2 RNA, herein nsp8, is amplified using RT-LAMP. The RT-LAMP products are scanned by the Cas12a-gRNA ribonucleoprotein (RNP) complex. The RNP binds to the specific complementary to gRNA, activating the transcleavage activity of Cas12a. The active Cas12a system cleaves a short ssDNA reporter that is labeled preferably, with a fluorophore and a quencher on either end. Cleavage of the reporter separates the quencher from the fluorophore, and fluorescence that is detectable with the naked eye is generated.