ToBRFV Viral Marker for Human Fecal Source Tracking
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Solution Overview
Problem
Current microbial indicators for human fecal contamination, such as total coliforms, Escherichia coli, and enterococci, are not exclusive to human sources and can be present in environmental samples, making it difficult to accurately assess human fecal contamination in water bodies.
Innovation Solution
The use of tomato brown rugose fruit virus (ToBRFV) as a marker for human fecal contamination through nucleic acid-based detection methods, including droplet digital PCR and quantitative PCR, to selectively amplify and detect ToBRFV sequences in samples, allowing for the quantification of human fecal contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional microbial indicators (total coliforms, E. coli, enterococci) are used to assess fecal contamination, then detection sensitivity is improved, but source specificity deteriorates because these indicators are found in both human and non-human feces
Solution Approach 1:
The patent segments the detection approach by using multiple viral indicators with different host specificities. Instead of relying on a single microbial indicator that detects all fecal sources, the invention divides the detection into human-specific viruses (ToBRFV, PMMoV) and omnivorous viruses (BCoV, HuNoV), allowing differentiation of contamination sources through specific primer sets for each virus type
Solution Approach 2:
The patent introduces viral RNA as an intermediary marker between fecal contamination and detection. Viruses serve as biological mediators that are more specific to human sources than traditional bacterial indicators, and their RNA can be selectively amplified using PCR techniques to determine the human versus animal origin of contamination
2Ease of operation
If traditional microbial indicators are used, then ease of detection is improved, but quantification accuracy deteriorates because these indicators cannot distinguish human fecal material from environmental sources
Solution Approach 1:
The patent creates a universal detection system that can identify multiple viral indicators in a single wastewater sample using PCR amplification. The multi-functionality allows the same basic detection platform to target different viruses (ToBRFV, PMMoV, BCoV, HuNoV) with different primer sets, providing both ease of operation and accurate human-specific quantification
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
ToBRFV provides a specific and sensitive method for detecting human fecal contamination, distinguishing it from non-human sources and enabling accurate quantitation of human fecal material in various samples, including wastewater and environmental samples.
Implementation Method 1
amplifying the nucleic acids using a set of primers capable of selectively amplifying at least a portion of the ToBRFV RNA
Implementation Method 2
detecting the presence of the amplified nucleic acids using a detectably labeled oligonucleotide probe sufficiently complementary to and capable of hybridizing with the ToBRFV RNA or amplicon thereof
Data Source
AI summary
Compositions and methods are provided for detecting the tomato brown rugose fruit virus (ToBRFV), which is highly abundant in human stool and wastewater. In particular, methods of using ToBRFV in microbial source tracking as a marker of human fecal contamination are disclosed. The methods utilize primers and probes capable of amplifying and/or detecting target ToBRFV sequences in samples suspected of having human fecal contamination. Methods of using ToBRFV in wastewater surveillance of pathogens as a marker to quantitate feces are also provided.