qPCR Viability Detection for Process-Relevant Microorganisms
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Solution Overview
Problem
Current methods for determining live cell numbers of process-relevant microorganisms in wastewater and biogas plants lack precision and speed, particularly in identifying active, process-disrupting microorganisms like nitrifiers and denitrifiers, which are crucial for efficient nitrogen degradation and process optimization.
Innovation Solution
The method employs quantitative real-time PCR (qPCR) with viability reagents like PMA or EMA to selectively quantify living cells by blocking dead cell DNA, ensuring only active microorganisms are counted, using a fluorescently labeled gene probe and Taq polymerase to amplify specific DNA sections, allowing for rapid and accurate analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If microscopy and/or FISH technology is used to detect bacteria, then the method is relatively simple to perform, but the precision and specificity are not particularly high and the testing methods are relatively time-consuming
Solution Approach 1:
The patent replaces mechanical/optical detection methods (microscopy and FISH technology) with a biochemical amplification method (quantitative real-time PCR). This substitution enables highly specific and precise detection of process-relevant microorganisms by targeting their DNA sequences, while significantly reducing the time required for analysis compared to traditional microscopy and FISH approaches.
2Reliability
If conventional PCR methods are used to amplify DNA, then the amplification process is established, but it cannot distinguish between living and dead cells and lacks viability information
Solution Approach 1:
The patent applies viability reagents (such as PMA or EMA) to the sample before performing PCR amplification. These reagents selectively penetrate dead cells and bind to their DNA, preventing amplification. Living cells exclude the reagents due to intact membranes, allowing their DNA to be amplified. This preliminary treatment step enables the subsequent PCR to specifically amplify DNA from viable cells only, providing reliable viability assessment without significantly increasing overall method complexity.
3Productivity
If quantitative real-time PCR with viability reagents is used, then high specificity and rapid results are achieved, but the method requires multiple components and steps
Solution Approach 1:
The patent combines multiple functions into an integrated quantitative real-time PCR assay with viability detection. The method merges DNA extraction, viability reagent treatment, PCR amplification with specific primers and fluorescent probes, and real-time quantification into a single workflow. This integration achieves high productivity through rapid automated processing while managing complexity through standardized protocols and ready-to-use reagent systems.
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 approach provides a rapid, cost-effective, and highly specific method for determining live cell numbers of process-relevant microorganisms, ensuring accurate assessment of microbiological processes and minimizing process disruptions in wastewater and biogas plants.
Implementation Method 1
an additional, target-gene-specific fluorescently labeled gene probe is used in addition to the two primers. During the amplification process, the gene probe is degraded, and the bound fluorophore is released. The resulting fluorescence signal, which increases with each cycle, quantifies the original target gene quantity in the initial sample.
Implementation Method 2
DNA polymerase to copy the defined section deoxyribonucleoside triphosphates as building blocks for the DNA strand synthesized by the DNA polymerase
Implementation Method 3
1. Denaturation: The double-stranded DNA is heated to separate the strands.
Implementation Method 4
2. Primer hybridization: The temperature is lowered to allow the primers to anneal to the single strand of DNA.
Data Source
AI summary
Method for determining the number of viable cells of process-relevant microorganisms in a sample, wherein the sample is examined using quantitative real-time PCR.