Microbiome Analysis Internal Standard Quantification
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Solution Overview
Problem
Current microbiome analysis methods face challenges in achieving uniform and reproducible DNA isolation, leading to inaccuracies in representing the composition of microbial communities due to variations in cell lysis and PCR amplification, which affect the quantification and relative abundance of bacterial species.
Innovation Solution
Incorporating a quantifiable standard of spike bacteria into the sample, which are added before DNA isolation and treated similarly to the microbiota, allows for absolute quantification and normalization of microbiome analysis, enabling process control and correction of methodological variances.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA isolation is performed using classic microbiological culture methods, then the analysis can be carried out, but more than 80% of the microbiota cannot be cultivated leading to incomplete representation of the microbial community
Solution Approach 1:
The patent replaces mechanical culture-based isolation methods with molecular genetic analysis methods (PCR amplification and sequencing of marker genes). This substitution allows detection of microorganisms that cannot be cultivated using traditional methods, thereby achieving complete representation of the microbial community including the 80% that are cultivation-resistant.
2Manufacturing precision
If DNA isolation is performed manually or using commercial kits, then DNA can be extracted, but uniform isolation from complex sample materials is problematic leading to non-standardizable inaccuracy in species distribution
Solution Approach 1:
The patent introduces an internal standard as an intermediary substance that is added to the sample before DNA isolation. This internal standard serves as a reference that allows quantification and normalization of the DNA extraction process, enabling detection and correction of deviations from uniform isolation, thereby improving both the standardizability and accuracy of species distribution measurements.
3Productivity
If PCR amplification is performed using commercially available DNA polymerases, then amplification can be carried out, but variations in amplification efficiency lead to non-reproducible results
Solution Approach 1:
The patent implements a feedback mechanism by adding an internal standard with known concentration to the PCR reaction. The amplification of the internal standard serves as a reference that allows real-time monitoring and normalization of amplification efficiency variations. This feedback enables correction of non-reproducible effects, ensuring consistent and reliable results across different experiments.
4Measurement precision
If relative quantification is used based on sequence data, then microbiome composition can be analyzed, but absolute quantification and process control are not possible
Solution Approach 1:
The patent introduces an internal standard as an intermediary reference substance with known absolute concentration. This internal standard is processed together with the sample through all steps (DNA isolation, PCR amplification, sequencing). By comparing the sequence reads of the internal standard against its known concentration, the system enables calculation of absolute quantification factors that can be applied to the sample data, thereby recovering the absolute quantification capability that was lost in relative analysis.
Data Source
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AI summary
The present invention relates to a method for microbiome analysis in which a quantifiable standard is added to a sample. The invention further relates to a device for carrying out a method for microbiome analysis using an internal standard. The invention also relates to a kit for carrying out a microbiome analysis.