Proteinase K-Free Nucleic Acid Extraction Buffer System
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Solution Overview
Problem
Current multiplex assays for detecting respiratory viruses, such as the ThermoFisher TaqPath COVID multiplex assay, face challenges including high background noise, poor stability of controls, increased labor costs due to RNA manipulation, and limited accessibility, which affect sensitivity and accuracy in detecting multiple viruses like SARS-CoV-2 and influenza viruses simultaneously.
Innovation Solution
A GRD buffer system is introduced, which is free of proteinase K and consists of a lysis buffer, wash buffer, and elution buffer, along with a method using a plasmid-based control and total human RNA for improved nucleic acid extraction and PCR, reducing background noise and increasing sensitivity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional multiplex PCR assays are used to detect multiple respiratory viruses, then the ability to detect multiple viruses simultaneously is achieved, but background noise increases and sensitivity decreases
Solution Approach 1:
The patent modifies the chemical composition parameters of the extraction buffer by incorporating specific chaotropic agents (guanidine hydrochloride, sodium chloride) and non-ionic detergents (Triton X-100, Tween 20) at optimized concentrations. These parameter changes enable effective nucleic acid extraction while minimizing background noise interference in multiplex PCR assays, thereby maintaining high sensitivity across multiple virus detections
2Productivity
If proteinase K is used in the extraction buffer, then nucleic acid extraction efficiency is improved, but the complexity of the buffer system increases and stability decreases
Solution Approach 1:
The patent removes proteinase K from the extraction buffer formulation while maintaining effective nucleic acid extraction. This is achieved by taking out the proteolytic enzyme component and relying instead on the synergistic action of chaotropic agents and detergents to disrupt viral structures and release nucleic acids, thereby simplifying the buffer system and improving stability without sacrificing extraction efficiency
3Reliability
If RNA manipulation steps are included in the assay protocol, then control stability is improved, but labor costs and processing time increase
Solution Approach 1:
The patent employs a self-service approach where the extraction buffer automatically performs multiple functions including viral lysis, nucleic acid release, and inhibition of nucleases through its built-in chaotropic agents and detergents. This eliminates the need for separate RNA manipulation steps such as additional purification or stabilization procedures, thereby maintaining control stability while reducing labor costs and processing time
4Measurement precision
If multiple washing steps are performed in PCR-based detection, then detection accuracy is improved, but time and cost for handling increase
Solution Approach 1:
The patent performs preliminary action by incorporating all necessary buffer components (chaotropic agents, detergents, salts) into the extraction buffer before the extraction process begins. This preliminary formulation ensures that nucleic acids are effectively released and protected from degradation in a single step, eliminating the need for multiple subsequent washing steps while maintaining detection accuracy and reducing handling time
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 GRD buffer system provides low background noise and higher sensitivity in detecting multiple respiratory viruses, including SARS-CoV-2 and influenza viruses, with reduced labor costs and improved stability of controls, enhancing the accuracy and accessibility of the testing process.
Implementation Method 1
the GRD-LB comprises a first buffer and a first chaotropic agent
Implementation Method 2
the GRD-WB comprises a second buffer, a second chaotropic agent, and an alcohol
Data Source
AI summary
A GRD buffer system, which is free of proteinase K, for extracting nucleic acid is provided. The GRD buffer system comprises a GRD lysis buffer (GRD-LB), a GRD wash buffer (GRD-WB), and a GRD elution buffer (GRD-EB). Also provided is a method of extracting nucleic acid with the above GRD buffer system.