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Publication Number
US-12480166-B2
Publication Date
2025-11-25
Expiration Date
Abstract
Compositions, testing chambers and methods for testing a fuel sample for microbial contamination (including fuels treated with a biocide) are provided, which comprise: a quantity of hydrocarbon fuel; a microbial contamination wherein the microbial contamination further comprises nucleic acid in the form of both DNA, RNA or a combination thereof, and an analyzing solution; wherein the analyzing solution comprises at least six (6) primer pairs for amplification of at least one target locus, wherein at least one primer of each pair of primers is labeled with a fluorescent dye and wherein at least one of the primer pair binds to the nucleic acid of the microbial contamination.
Core Innovation
The compositions comprise fluorescently labeled primer pairs for multiplex PCR-based detection and identification of microbial contamination in hydrocarbon samples. These compositions comprise either at least six or at least twelve primer pairs, wherein at least one primer of each pair is labeled with a fluorescent dye and exhibits at least 90% homology to designated sequences [procedural detail omitted for safety]. Assays target bacterial and archaeal 16S, fungal and yeast 18S, internal transcribed spacer regions, and functional loci including dsrB and mcrA.
The disclosure describes Rapid DNA processing, microfluidic amplification, and electrophoresis for direct analysis of hydrocarbon samples without fuel-water phase separation. Workflows evaluate fragment traces, electropherogram peaks, and internal controls to generate full- or partial-profile outputs across bacterial, archaeal, fungal, and yeast targets. Documented evaluations include jet fuel, diesel, and gasoline samples to support contamination tracing, biocide efficacy assessment, low-copy RNA-transcript detection, and fuel prospecting.
Claims Coverage
The patent claims identify two independent claims covering compositions of fluorescently labeled primer pairs across two inventive features.
Composition with at least six fluorescently labeled primer pairs
The claim element recites a composition comprising at least six primer pairs, wherein at least one primer of each pair is labeled with a fluorescent dye, and the primers have at least 90% homology to designated sequences [procedural detail omitted for safety].
Composition with at least twelve fluorescently labeled primer pairs
The claim element recites a composition comprising at least twelve primer pairs, wherein at least one primer of each pair is labeled with a fluorescent dye, and the primers have at least 90% homology to designated sequences [procedural detail omitted for safety].
The two independent claims cover fluorescently labeled primer-pair compositions defined by minimum panel sizes of six and twelve primer pairs with specified sequence-homology requirements, while dependent claims further recite sequence homology thresholds, dye use, primer features, composition form, and specific targets.
Stated Advantages
The assays support direct analysis of fuel samples without fuel-water phase separation.
The description reports detection of Bacillus cereus in varied fuels at low copy inputs.
The description presents field-deployable Rapid DNA processing for sample analysis.
Documented Applications
Multiplex detection and mapping of assay targets across bacterial, archaeal, fungal, and yeast panels, including Bacillus, Pseudomonas, Penicillium, Aspergillus, and Candida, using ribosomal, internal transcribed spacer, and non-ribosomal targets.
Fuel-sample assays in jet fuel, diesel, and gasoline, targeting bacterial 16S, archaeal 16S, fungal and yeast 18S, sulfate-reduction dsrB, and methanogenesis mcrA for detection and identification of microbial contamination.
Fuel prospecting and prospecting for methane-, ethene-, propane-, and butane-oxidizing microorganisms using designated gene targets.
Contamination tracing and assessment of biocide efficacy.
RNA-transcript detection, including representative traces documenting low-copy detection.
Target analysis and allele assignment, including targets pycA, ilvD, pta, gmk, tpi, and glpF, with workflows producing full- or partial-profile outputs.
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