Species-specific primer sets and identification of species-specific DNA sequences using genome fragment enrichment
Inventors
Shanks, Orin C. • Domingo, Jorge Santo • Graham, James E. • Lu, Jingrang
Assignees
US Environmental Protection Agency
Publication Number
US-8574839-B2
Publication Date
2013-11-05
Expiration Date
2025-12-27
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Abstract
Targeted sequencing of genetic regions that differ between two DNA preparations uses genomic fragment enrichment. This method can be used to study genetic variation among closely related species and microbial communities, particularly for identifying sources of fecal pollution.
Core Innovation
The invention provides a method called Genome Fragment Enrichment (GFE) which identifies microbial DNA sequences specific to particular species or microbial communities, enabling determination of different sources of fecal contamination. It uses a competitive solution phase hybridization approach to enrich genomic regions that differ between two DNA preparations, such as microbial metagenomes from fecal samples of different animals, allowing for the selection and amplification of source-specific DNA sequences. These sequences can be used to develop species-specific PCR primer sets to differentiate among bacterial species, strains, and pollution sources.
The problem addressed by the invention is the challenge in accurately detecting and discriminating sources of fecal pollution in environmental waters, especially poultry fecal contamination, due to limitations of existing methods. Current regulatory methods using culturable fecal indicator bacteria lack specificity to differentiate among bacterial strains or animal sources. Previous genotypic methods reliant on 16S rDNA sequences or culturing face limitations related to secondary habitat populations, geographic variability, and inability to target microbial genes involved in host-microbe interactions, which are expected to provide higher genetic variation specific to respective hosts.
The invention overcomes these challenges by applying GFE to isolate DNA fragments unique or divergent to particular animal fecal microbial communities, including chicken, cow, and human sources. The method provides a positive selection technique enriching for DNA sequences specific to one source relative to another, enabling development of highly specific PCR assays. These assays demonstrate specificity and broad geographic distribution among target hosts, and can detect fecal contamination in environmental water samples, thereby improving microbial source tracking and environmental water quality assessment.
Claims Coverage
The patent contains multiple independent claims covering methods for identifying sources of fecal contamination using PCR primers identified by genome fragment enrichment, and the genome fragment enrichment process itself. Key inventive features are extracted below.
Method for identifying source-specific DNA sequences via genome fragment enrichment
A method for detecting a fecal contamination source by providing microbial community DNA from fecal contamination in water, amplifying source-specific genomic DNA using PCR primers identified by genome fragment enrichment (GFE), and detecting the PCR product. The GFE process involves hybridizing labeled genomic DNA from a first microbial species with PCR-blocked genomic DNA from a second species, self-hybridizing with terminal-tagged amplified fragments from the first species, and isolating resulting DNA hybrids to obtain source-specific sequences.
Use of chicken-specific primer sets for detecting chicken fecal contamination
Using at least one chicken-specific PCR primer pair or combination, identified by GFE, to amplify and detect chicken-specific microbial community DNA in fecal contamination samples, with the primers selected from defined chicken-specific primer sets.
Use of bird-specific primer sets for detecting bird fecal contamination
Using bird-specific PCR primer pairs or combinations, identified by GFE, to amplify and detect bird-specific microbial community DNA in fecal contamination samples.
Applying GFE with composite DNA pools for enhanced source discrimination
Performing GFE using composite DNA pools as tester and blocker to enrich for source-specific DNA fragments, improving representation of host metagenomes and reducing assay cross-reactivity.
Genome fragment enrichment method
A method comprising providing labeled sheared total genomic DNA from a first microbial species and PCR-blocked sheared genomic DNA from a second species; prehybridizing these DNAs; self-hybridizing with PCR-amplified fragments bearing defined terminal sequence tags from the first species; and isolating DNA hybrids, enabling enrichment of DNA sequences unique to the first species.
Use of multiple primers recognizing diverse microorganisms from a source
Using a plurality of PCR primers specific to a source in amplification to collectively recognize DNA from multiple different microorganisms within that source's microbial community.
The claimed invention covers a method for detecting source-specific fecal contamination using PCR primers developed via genome fragment enrichment, including specific applications to chicken and bird fecal sources, with the GFE method encompassing competitive hybridization steps to enrich unique DNA fragments from complex microbial communities. The claims also cover use of composite DNA pools and multiple primers to enhance source differentiation.
Stated Advantages
The method allows rapid identification of numerous species- or strain-specific DNA sequences useful as genetic markers for fecal source tracking.
GFE provides a positive physical selection approach, reducing false positives compared to PCR-mediated subtractive hybridization methods.
Host-specific PCR assays developed show high specificity and broad geographic and temporal stability among target animal populations.
The approach enables detection of host-specific microbial sequences absent or divergent in closely related species, improving discrimination over existing 16S rDNA methods.
The technique facilitates development of PCR and real-time PCR assays, as well as potential microarray applications for environmental monitoring and risk assessment.
Documented Applications
Detection and identification of chicken-specific fecal contamination in environmental water samples.
Differentiating among fecal contamination sources from cattle, human, pig, chicken, turkey, goose, seagull, and other animals in watersheds.
Development of species-specific PCR primers for microbial source tracking (MST) in compliance with environmental regulations such as the U.S. Clean Water Act and Total Maximum Daily Loads (TMDLs).
Application in environmental water quality assessment, recreational water safety, and drinking water source protection.
Identification of microorganism-specific genetic markers relevant to virulence, stress response, metabolism, and host-microbe interactions.
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