Double-Stranded RNA Fragmentation for Unknown Virus Detection
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Solution Overview
Problem
Conventional virus detection methods are limited by the need for specific antibodies or nucleic acid primers, making it difficult to detect unknown viruses and are inefficient in separating viral sequences from non-viral cell-derived sequences, leading to biased results.
Innovation Solution
A method involving the random fragmentation of double-stranded RNA (dsRNA) using ultrasonication, followed by reverse transcription and PCR, to obtain DNA fragments for sequence analysis, allowing for the determination of full-length genome sequences of RNA viruses, including unknown ones.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional virus detection methods using specific antibodies or nucleic acid primers are used, then detection of known viruses is achieved, but detection of unknown viruses is limited
Solution Approach 1:
The method extracts and enriches dsRNA from complex viral samples before sequencing. Since dsRNA is characteristic of many viruses but rare in host cells, this extraction step isolates viral genetic material from overwhelming non-viral sequences, enabling detection of both known and unknown viruses without requiring prior sequence information
Solution Approach 2:
The dsRNA enrichment approach serves multiple functions: it concentrates viral genetic material, removes the need for virus-specific primers or antibodies, and enables simultaneous detection of diverse virus types including those with unknown sequences. This universal method replaces multiple virus-specific detection methods with a single versatile protocol
2Manufacturing precision
If physical disruption methods are used for DNA fragmentation, then random fragmentation without bias is achieved, but the method does not address phosphate group presence at cleavage ends needed for adapter addition
Solution Approach 1:
The patent introduces T4 polynucleotide kinase as an intermediary enzyme that acts on the fragmented dsRNA ends. This enzyme transfers phosphate groups from ATP to the 5' hydroxyl ends of the fragments, ensuring all fragments have the necessary phosphate groups for adapter ligation while preserving the random fragmentation pattern achieved by physical disruption
Solution Approach 2:
The method changes the chemical state of fragment ends by adding phosphate groups through enzymatic action. This parameter change (from hydroxyl to phosphate) enables subsequent adapter addition while maintaining the benefits of random physical fragmentation
3Quantity of substance
If comprehensive sequencing of all nucleic acid in samples is performed, then all viral sequences are captured, but viral sequences are lost among overwhelming non-viral cell-derived sequences
Solution Approach 1:
The method extracts dsRNA specifically from total nucleic acid samples. Since dsRNA is abundant in viruses but rare in host cells, this extraction enriches viral sequences while removing overwhelming non-viral DNA and RNA, dramatically improving the proportion of viral sequences in the final data set
Solution Approach 2:
The approach changes the quality composition of the nucleic acid sample by selecting for dsRNA molecules. This local quality change (from mixed nucleic acid types to dsRNA-enriched material) concentrates viral genetic material while excluding most host cell sequences, improving detection efficiency without sacrificing comprehensive viral coverage
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 efficient and uniform sequencing of long to short dsRNA molecules, including sequences around the ends of RNA genomes, improving the detection of RNA viruses and identifying novel virus sequences.
Implementation Method 1
the objective dsRNA is mechanically fragmented by ultrasonication
Data Source
Figure 1
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Figure 3a~3d
AI summary
The object is to provide a method that enables detection of unknown virus sequences and efficient detection and search of viruses. The method comprises the step of randomly fragmenting an objective double-stranded (ds) RNAto obtain dsRNA fragments; the step of subjecting the obtained dsRNA fragments to a reverse transcription reaction and then performing polymerase chain reaction (PCR) to obtain corresponding DNA fragments; and the step of subjecting the obtained DNA fragments to a sequence analysis operation to determine the sequences. The reverse transcription reaction is preferably started from the 3' ends of the dsRNA fragments.