Ligation-Assisted RNA Gene Detection Without Reverse Transcription
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Solution Overview
Problem
Existing isothermal amplification methods for viral RNA detection, such as LAMP, suffer from low selectivity due to the use of reverse transcriptase, leading to false-positive and false-negative diagnoses, especially for RNA viruses like SARS-CoV-2, as they struggle to distinguish between similar viral RNAs and human genome sequences.
Innovation Solution
A dual-site ligation-assisted cDNA synthesis method that does not use reverse transcriptase, employing a ligase and multiple DNA oligonucleotide template sequences to link template sequences, followed by LAMP reaction, ensuring cDNA synthesis only occurs in the presence of the target RNA, thereby enhancing selectivity and allowing rapid detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If reverse transcriptase is used in LAMP for viral RNA detection, then amplification can be performed, but selectivity decreases leading to false-positive and false-negative diagnoses
Solution Approach 1:
The invention extracts and removes reverse transcriptase from the LAMP detection system. Instead of using reverse transcriptase to synthesize cDNA from viral RNA, the patent employs a ligation-based approach where template sequences are ligated to form a template for LAMP amplification, thereby eliminating the selectivity problems associated with reverse transcription while maintaining amplification capability
Solution Approach 2:
The invention introduces a ligation step as an intermediary between RNA detection and LAMP amplification. Template sequences complementary to the target RNA are ligated together to form a double-stranded DNA template, which then serves as the substrate for LAMP amplification. This intermediary step provides the necessary selectivity through specific ligation while enabling subsequent robust amplification
2Measurement precision
If primer sequence is excessively specific to avoid false positives, then selectivity improves, but binding ability decreases leading to false-negative detection
Solution Approach 1:
The invention segments the detection specificity requirement into multiple separate template sequences rather than relying on a single highly specific primer. Each template sequence targets a different region of the viral RNA, and all must be successfully ligated for amplification to occur. This segmentation allows each individual template to have moderate specificity while the combination provides high overall selectivity without sacrificing sensitivity
Solution Approach 2:
The invention performs preliminary ligation of multiple template sequences to form a complete double-stranded DNA template before initiating LAMP amplification. This preliminary action ensures that all necessary specific binding events occur before amplification begins, creating a robust template that guarantees both selectivity (through multiple specific ligation events) and sensitivity (through the cooperative effect of all templates being present)
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 enables rapid, sensitive, and selective detection of target genes, such as SARS-CoV-2 RNA, with visual confirmation of results in about 30 minutes at room temperature, suitable for point-of-care genetic molecular diagnosis.
Implementation Method 1
a dual-site ligation-assisted cDNA synthesis method that does not use reverse transcriptase, employing a ligase and multiple DNA oligonucleotide template sequences to link template sequences
Implementation Method 2
loop-mediated isothermal amplification (LAMP)
Data Source
AI summary
The present invention relates to a composition for detecting a target gene based on cDNA synthesis using a ligation method that does not use reverse transcription and a method for multiple ligation-assisted recombinase polymerase amplification, and since a target gene may be detected through a visual change with only a short reaction time of about 30 minutes at room temperature without the synthesis of cDNA using reverse transcriptase, the present invention may be effectively used for point-of-care genetic molecular diagnosis of RNA viruses and the like.