Bacterial Concentration Detection via Isothermal Amplification and MPN
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Solution Overview
Problem
Traditional methods for determining the concentration of bacteria like Salmonella spp. and Legionella spp. in environmental matrices are laborious, time-consuming, require large amounts of materials, and pose biological risks, making them inefficient and costly.
Innovation Solution
A method involving the preparation of primary suspensions, dilutions, genome extraction, and isothermal amplification for qualitative analysis, followed by quantitative analysis using the MPN technique to determine bacterial concentration, reducing the need for extensive culture growth and minimizing material usage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional culture-based methods are used to determine bacterial concentration, then reliable detection of Salmonella spp. and Legionella spp. is achieved, but analysis time becomes excessively long (6-14 days) and material consumption increases
Solution Approach 1:
The traditional single culture process is segmented into two independent detection methods: (1) culture-based methods for reliable detection, and (2) molecular biology methods (PCR, LAMP, RNA-seq) for rapid identification. This segmentation allows the system to obtain results from both reliable culture methods and fast molecular methods, reducing overall analysis time while maintaining detection reliability.
Solution Approach 2:
Molecular biology techniques serve as an intermediary that bridges the gap between slow culture methods and the need for rapid results. By extracting DNA/RNA from cultured samples and applying PCR or LAMP amplification, the system accelerates the detection process without compromising the reliability established by prior culture steps.
2Measurement precision
If traditional culture methods are used, then accurate bacterial identification is achieved, but material consumption and disposal costs increase significantly
Solution Approach 1:
The invention extracts only the essential genetic material (DNA or RNA) from bacterial samples for analysis, rather than requiring large volumes of culture media and reagents. This extraction approach reduces material consumption while maintaining identification accuracy through targeted molecular analysis of extracted nucleic acids.
Solution Approach 2:
Molecular biology techniques create copies of specific genetic sequences through PCR or LAMP amplification, allowing analysis of minute amounts of extracted DNA/RNA. This copying process eliminates the need for large quantities of original sample material while preserving measurement precision through repeated amplification of target sequences.
3Reliability
If traditional culture methods are employed, then comprehensive bacterial analysis is achieved, but biological risk to operators increases
Solution Approach 1:
The invention replaces mechanical handling of live cultures with molecular biology techniques that work on extracted DNA or RNA. By substituting the mechanical culture process with in vitro molecular amplification and detection, the system maintains analysis completeness while eliminating direct exposure to pathogenic bacteria, thereby reducing biological risk to operators.
4Quantity of substance
If extensive culture growth is performed, then sufficient bacterial material for analysis is obtained, but analysis time and operational complexity increase
Solution Approach 1:
The invention performs preliminary extraction of DNA or RNA from bacterial samples before amplification and analysis steps. This preliminary action prepares the genetic material in advance, allowing subsequent PCR or LAMP reactions to proceed rapidly without requiring extensive culture growth, thereby reducing both analysis time and operational complexity.
Solution Approach 2:
The invention changes the fundamental parameter of detection from measuring bacterial mass through culture growth to detecting specific genetic sequences through molecular amplification. This parameter change allows analysis to proceed with minimal initial bacterial material, reducing the need for extensive culture conditions and simplifying the overall operational process.
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 method significantly reduces analysis time, material consumption, and biological risk while providing accurate results in under 24 hours, saving costs and logistical challenges.
Implementation Method 1
Applying at least one qualitative analysis procedure to each tube, said qualitative analysis procedure being configured for detecting the presence or absence of the genome of said bacterium of interest
Implementation Method 2
Applying a quantitative analysis procedure, said quantitative analysis procedure being configured to determine the value of the most probable number of said bacteria of interest in said at least one sample of environmental matrix
Data Source
Figure 1
Figure 2
Figure 3a~3c
AI summary
Method (1) for determining the concentration of a bacterium of interest in one environmental matrix, the method comprising the following operational steps: A. Preparing at least one primary suspension (2) of one sample of the environmental matrix, thereby obtaining the breaking down of the particles of the environmental matrix and the separation of the bacterium of interest, if present, of the environmental matrix; B. Preparing one or more dilutions (21, 22, 23) of the primary suspension (2) in respective first containers (31, 32, 33); C. Preparing second containers (401, 402, 403; 411, 412, 413; 421, 422, 423; 431, 432, 433; 441, 442, 443) containing the at least one primary suspension (2) and said one or more dilutions (21, 22, 23); D. extracting the genome of the content of each second container (401, 402, 403; 411, 412, 413; 421, 422, 423; 431, 432, 433; 441, 442, 443) into a respective third container (501, 502, 503; 511, 512, 513; 521, 522, 523; 531, 532, 533; 541, 542, 543); E. Preparing, at least one plurality of tubes (601, 602, 603; 611, 612, 613; 621, 622, 623; 631, 632, 633; 641, 642, 643), each tube of the plurality of tubes containing the genome contained in a respective third container (501, 502, 503; 511, 512, 513; 521, 522, 523; 531, 532, 533; 541, 542, 543); F. Applying at least one qualitative analysis procedure to each tube (601, 602, 603; 611, 612, 613; 621, 622, 623; 631, 632, 633; 641, 642, 643), the qualitative analysis procedure being configured for detect the presence or absence of the genome of the bacterium of interest, and outputting for each tube (601, 602, 603; 611, 612, 613; 621, 622, 623; 631, 632, 633; 641, 642, 643) a corresponding index (101, 102, 103; 111, 112, 113; 121, 122, 123; 131, 132, 133; 141, 142, 143) of the presence or absence of the genome of the bacterium of interest; G. Based on each index (101, 102, 103; 111, 112, 113; 121, 122, 123; 131, 132, 133; 141, 142, 143) of the presence or absence of the genome of the bacterium of interest, applying a quantitative analysis procedure, the quantitative analysis procedure being configured to determine the value of the most probable number of said bacteria of interest in the at least one sample of environmental matrix.