Host-associated DNA sequences, primers, and probes for PCR-based identification of dog fecal pollution sources
Inventors
Shanks, Orin C. • GREEN, Hyatt C.
Assignees
US Environmental Protection Agency • Government of the United States of America
Publication Number
US-9816141-B2
Publication Date
2017-11-14
Expiration Date
2034-03-31
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Abstract
A method for detecting dog-fecal contamination in a sample, comprising assaying the sample using a nucleotide sequence based genetic assay which comprises contacting the sample with at least one nucleic acid molecule having the nucleic acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, the nucleic acid sequence being capable of binding to a nucleic acid sequence in the sample, and detecting binding of the nucleic acid molecule to the nucleic acid sequence in the sample, wherein a presence of binding is indicative of the presence of dog-fecal contamination in the sample; the nucleic acid molecules; and a kit comprising at least two of the above-described nucleic acid molecules.
Core Innovation
The invention provides a method for detecting dog-fecal contamination in a sample by assaying the sample with a nucleotide sequence based genetic assay that uses nucleic acid molecules having sequences shown in SEQ ID NOs: 1 through 11 or their complements. These nucleic acid molecules bind to specific nucleic acid sequences present in the sample, and the detection of such binding is indicative of the presence of dog-fecal contamination.
The problem solved arises from the difficulty of discriminating fecal contamination sources based on current microbial water quality assessment methods, which typically rely on culturable fecal indicator bacteria such as Enterococci and other coliforms. These methods cannot distinguish specific bacterial strains or animal sources. Existing microbial source tracking methods targeting genetic material, often based on 16S rRNA gene sequences, face limitations due to lack of specificity, especially at the species level, and the presence of non-gut secondary habitat populations. Moreover, prior techniques have not targeted microorganism DNA sequences encoding proteins directly involved in host-microbe interactions, which possess higher genetic variation related to host specificity.
The invention addresses these deficiencies by providing nucleic acid molecules targeting non-ribosomal genes predicted to be involved in host-associated metabolic activities specific to dogs. This novel approach allows for differentiation between dog fecal pollution and other animal fecal sources. Additionally, the method and kits described have been developed to be effective across different dog populations throughout the United States, overcoming geographic limitations encountered by previous methods.
Claims Coverage
The patent contains one independent claim defining the core method for detecting dog-fecal contamination, which encompasses key inventive features relating to the nucleic acid sequences used and detection methods.
Use of specific dog-associated nucleic acid sequences for detection
The method involves contacting a water sample with at least one nucleic acid sequence chosen from SEQ ID NOs: 1 through 11 or their fully complementary sequences to specifically bind dog-fecal contamination related genetic material.
Detection of binding as evidence of dog-fecal contamination
The presence of binding between the nucleic acid molecules and sequences in the sample is used as evidence indicative of dog-fecal contamination.
Use of amplification reactions for detection
The method includes detecting binding via amplification reactions such as PCR, quantitative PCR (qPCR), or digital PCR to identify dog-fecal bacteria in the water sample.
The claims cover a method for detecting dog-fecal contamination by using specific nucleic acid sequences that bind to target DNA in samples, and employing amplification-based detection techniques to confirm the presence of contamination, enabling specific identification of dog fecal sources.
Stated Advantages
The method can differentiate between canine fecal pollution and other animal fecal contamination sources.
It targets non-ribosomal genes predicted to be involved in host-associated metabolic activities, which have not been previously used for host-associated fecal technologies.
The method is applicable across dog populations throughout the United States, demonstrating broad geographic utility compared to prior technologies.
Documented Applications
Detecting dog-fecal contamination in water samples for environmental and water quality monitoring.
Use in microbial source tracking to identify fecal pollution sources in recreational waters, urban stormwater runoff, sewage influent, and fecal sample analysis.
Inclusion in kits comprising nucleic acid molecules for testing presence of dog fecal contamination in environmental samples.
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