Viral Sample Preservation Solution for Direct qPCR
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Solution Overview
Problem
Current viral RNA detection methods are cumbersome, complex, and prone to errors, requiring separate kits for sample preservation and nucleic acid extraction, and are inefficient, especially in emergency situations like the SARS-CoV-2 outbreak, due to the need for stringent biosafety standards and refrigerated storage.
Innovation Solution
A virus preservation solution and nucleic acid amplification reaction preparation that allow for direct qPCR amplification without extraction, using a pH-buffered solution with bovine serum albumin, fish gelatin, and inhibitors, combined with M-MLV Reverse Transcriptase and Taq DNA polymerase, which can maintain sample stability at room temperature and enable rapid detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional separate kits for sample preservation and nucleic acid extraction are used, then sample stability is maintained, but the detection process becomes complex and time-consuming
Solution Approach 1:
The patent combines the sample preservation function and nucleic acid extraction function into a single integrated kit. The preservation solution contains not only stabilizing agents (BSA, fish gelatin) but also nucleic acid extraction reagents and PCR inhibitors, eliminating the need for separate extraction kits and reducing procedural complexity while maintaining sample stability.
Solution Approach 2:
The preservation solution is designed to perform multiple functions simultaneously: it preserves viral samples at room temperature, prevents RNA degradation, inhibits contaminating nucleases, and facilitates direct nucleic acid extraction. This multi-functional design reduces the number of steps and reagents needed in the detection workflow.
2Reliability
If refrigerated storage conditions (2-8°C or -20°C) are used for preservation solutions and PCR reagents, then sample stability is maintained, but logistics and operational complexity increase
Solution Approach 1:
The patent changes the temperature parameter from refrigerated (2-8°C) or frozen (-20°C) storage to room temperature storage. This is achieved by formulating the preservation solution with stabilizing agents (BSA at 1-10 μg/μl, fish gelatin at 0.1-2%) that maintain sample integrity at higher temperatures, and by adjusting the pH to 8.0-8.5 which optimizes both stability and enzyme activity across a broader temperature range.
3Reliability
If multiple separate reagent kits are used for preservation, extraction, and PCR, then each function is optimized, but the overall detection time increases to many hours
Solution Approach 1:
The patent merges preservation, extraction, and PCR amplification into a single-tube reaction system. The preservation solution contains all necessary components including nucleic acid extraction reagents and PCR reagents, allowing the entire process to occur in one tube without intermediate transfer steps, thereby reducing detection time from hours to minutes while maintaining accuracy.
Solution Approach 2:
The preservation solution is prepared in advance with all necessary components for subsequent PCR amplification. The pH is pre-adjusted to 8.0-8.5 and stabilizing agents are pre-added, so that when the sample is collected, it is already primed for direct amplification without requiring separate preparation steps, thus accelerating the overall detection process.
4Productivity
If viral samples are directly mixed with PCR reagents without extraction, then detection speed increases, but RT-PCR polymerase inhibitors in samples hinder amplification
Solution Approach 1:
The patent introduces fish gelatin and BSA as intermediary substances that bind to RT-PCR polymerase inhibitors present in viral samples. These intermediaries sequester the inhibitors, preventing them from interfering with the polymerase activity, thereby enabling direct amplification of viral nucleic acid from crude samples without extraction while maintaining high amplification efficiency.
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 solution enables rapid, high-sensitivity virus detection within 15-30 minutes, reducing the complexity and error probability of traditional methods and improving response times in emergency situations.
Implementation Method 1
The virus preservation solution has a pH in the range of between 8.0 to 8.5, for example, pH 8.0. The pH buffer can comprise Tris-HCl.
Implementation Method 2
The virus preservation solution comprises bovine serum albumin (BSA), fish gelatin, a surfactant... The virus preservation solution can preserve a viral sample and prevent RNA degradation at room temperature for an extended period of time.
Implementation Method 3
The nucleic acid amplification reaction solution can further comprise polyoxyethylene (20) sorbitan monolaurate. There is also provided a lyophilized powder prepared by lyophilizing the nucleic acid amplification reaction solution(s) described herein.
Implementation Method 4
The nucleic acid amplification reaction solution comprises M-MLV Reverse Transcriptase (MMLV), Taq DNA polymerase, dNTP, primers and fluorescence probes.
Implementation Method 5
The nucleic acid amplification reaction solution comprises M-MLV Reverse Transcriptase (MMLV), Taq DNA polymerase, dNTP, primers and fluorescence probes.
Data Source
AI summary
The present invention provides a rapid and high-sensitivity method to detect virus by qPCR nucleic acid amplification. A viral sample containing a RNA virus such as a corona virus is mixed with a virus preservation solution and a nucleic acid amplification reaction preparation (which can be in a lyophilized powder) to prepare the PCR-sample solution. The virus preservation solution can preserve the viral sample and prevent RNA degradation at room temperature for an extended period of time. No viral nucleic acid extraction step is needed.