Protease-Assisted Clinical Sample Preparation for Nucleic Acid Amplification
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Solution Overview
Problem
Nucleic acid amplification techniques in clinical samples are inhibited by common additives such as sodium polyanethol sulfonate (SPS) and heparin, leading to reduced detection sensitivity and efficiency in identifying pathogenic microbes.
Innovation Solution
The addition of proteases to clinical samples allows for the removal of polyanionic polymer nucleic acid amplification inhibitors by degrading the protein complexes they form, thereby enhancing nucleic acid amplification and detection sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If polyanionic detergents (e.g., SPS, heparin) are added to clinical samples to inhibit bactericidal processes and promote microbial survival, then microbial preservation is improved, but nucleic acid amplification is inhibited
Solution Approach 1:
The method separates the sample into different fractions through centrifugation, isolating the inhibitory polyanionic compounds from the nucleic acid-containing fraction. This physical segmentation removes the harmful interaction between SPS/heparin and the nucleic acid amplification process while preserving the microbial targets.
Solution Approach 2:
The inhibitory polyanionic compounds (SPS, heparin) are extracted and removed from the clinical sample through centrifugation and fractionation. By taking out these harmful substances, the nucleic acid amplification can proceed without inhibition while the microbial targets remain intact in the retained fraction.
2Object-affected harmful factors
If nucleic acid samples are diluted 10× to 1000× to mitigate SPS inhibition, then amplification inhibition is reduced, but nucleic acid detection sensitivity is significantly reduced
Solution Approach 1:
The method converts the harmful effect of SPS/heparin into a beneficial separation mechanism. The polyanionic compounds' tendency to interact with proteins and nucleic acids is exploited to partition them into different fractions during centrifugation, allowing the inhibitory substances to be removed while concentrating the nucleic acid targets in the retained fraction for enhanced sensitivity.
Solution Approach 2:
By segmenting the sample into supernatant and retained fraction through centrifugation, the method separates inhibitory compounds from nucleic acid targets. This physical division eliminates the need for dilution while removing inhibition, as the nucleic acids remain concentrated in the retained fraction without the harmful polyanionic substances.
3Speed
If rapid identification of pathogenic microbes is achieved through nucleic acid amplification, then diagnostic speed is improved, but detection accuracy is reduced due to additive inhibition
Solution Approach 1:
The method performs preliminary fractionation and removal of inhibitory substances before nucleic acid amplification. By pre-processing the sample to eliminate SPS and heparin through centrifugation-based separation, the subsequent amplification step can proceed with high accuracy without interference, maintaining both speed and detection precision.
Solution Approach 2:
The centrifugation process acts as an intermediary step between sample collection and nucleic acid amplification. This intermediate fractionation step separates and removes inhibitory compounds, creating a clean template for amplification that maintains both rapid processing and high detection accuracy.
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 improves nucleic acid amplification efficiency by 5-fold to 100-fold, enabling the detection of microbial nucleic acids at concentrations between 1 and 100,000 colony forming units/mL, thus facilitating rapid identification of pathogenic microbes.
Implementation Method 1
adding proteases to clinical samples reduces contamination by certain nucleic acid amplification inhibitors in processed clinical samples without otherwise substantially harming the integrity or quality of the samples
Data Source
AI summary
The present disclosure is a method of preparing a clinical sample comprising host cells, microbes, proteins and a polyanionic polymer nucleic acid amplification inhibitor.