Fluorescent E. coli Detection of Bacteriolytic Conditions
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Solution Overview
Problem
Current methods for detecting somatic coliphages in water and food samples are laborious, time-consuming, and suffer from low sensitivity and high false positive rates, limiting their practicality for rapid and accurate fecal pollution monitoring.
Innovation Solution
A method using a genetically modified E. coli strain that overexpresses the uidA gene with disrupted uidB and/or uidC genes, which contacts a substrate that undergoes a detectable change when cleaved by the enzyme, allowing for sensitive and specific detection of bacteriolytic conditions, including phage presence, without requiring sample concentration or long enrichment periods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard ISO 10705 methods are used for detecting somatic coliphages, then measurement precision is improved, but loss of time increases and productivity decreases
Solution Approach 1:
The invention changes the substrate parameter from non-fluorescent to fluorescent (MUG substrate), enabling direct fluorescent detection without waiting for colony formation. This parameter change in the detection system allows results to be obtained in 3.5-4.5 hours while maintaining measurement precision comparable to standard ISO 10705 methods.
2Loss of time
If Fast Phage method is used for rapid detection, then loss of time is reduced, but measurement precision deteriorates due to low sensitivity and high false positive rates
Solution Approach 1:
The invention uses a fluorescent substrate (MUG) as an intermediary that is cleaved by beta-glucuronidase released from lysed E. coli cells. This intermediary provides a direct fluorescent signal that is both sensitive and specific, eliminating the false positive problems of the Fast Phage method while maintaining rapid detection capability.
Solution Approach 2:
The invention changes the detection parameter from colorimetric (Fast Phage) to fluorescent detection. This parameter change enables highly sensitive and specific detection of low phage titers without the false positive issues that plague the Fast Phage method, while maintaining the rapid 3.5-4.5 hour detection time.
3Measurement precision
If multiple-step ISO protocol is used, then measurement precision is improved, but device complexity increases and ease of operation decreases
Solution Approach 1:
The invention merges the replication step and detection step into a single combined procedure. E. coli cells are incubated with the sample and fluorescent substrate simultaneously, allowing phage replication and enzyme release to occur together, followed by direct fluorescent detection. This merging eliminates the separate spotting and colony formation steps of the ISO protocol, reducing complexity while maintaining precision.
4Measurement precision
If sample concentration is performed to improve sensitivity, then measurement precision is improved, but loss of time increases and device complexity increases
Solution Approach 1:
The invention changes the detection parameter to fluorescent measurement, which provides sufficient sensitivity without requiring sample concentration. The fluorescent signal from MUG cleavage by beta-glucuronidase is detectable at very low concentrations, eliminating the time-consuming concentration steps while maintaining high detection sensitivity.
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 achieves sensitivity and specificity comparable to standard ISO 10705 methods in 3.5 to 4.5 hours, enabling detection of as few as 1 phage and reducing false positives, making it suitable for point-of-use, cost-effective, and rapid detection of fecal pollution indicators.
Implementation Method 1
contacting the test sample with a bacterial strain and a substrate which undergoes a detectable change when cleaved by a specific enzyme of said bacterial strain
Implementation Method 2
substrate is glucuronide linked by a glycosidic bond to a colorimetric or fluorimetric moiety
Implementation Method 3
substrate is glucuronide linked by a glycosidic bond to a colorimetric or fluorimetric moiety
Implementation Method 4
bacterial strain is E. coli overexpressing uidA gene and comprising disrupted uidB and/or uidC genes
Data Source
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AI summary
The present invention relates to a method for detecting bacteriolytic conditions in a test sample comprising the step of contacting the test sample with a bacterial strain and a substrate which undergoes a detectable change when cleaved by a specific enzyme of said bacterial strain, wherein the bacterial strain is unable of: (a) uptaking the substrate from the medium and (b) secreting the enzyme specific for the substrate outside of the cell and wherein detecting a change in the test sample due to the cleavage of the substrate by its specific bacterial enzyme indicates that bacteriolytic conditions exist in the test sample. The invention further provides a kit for performing the method of the invention.