Multiplexed Fuel Analysis
AssigneeANDE Corporation
InventorsRichard F. Selden, Rosemary Turingan Witkowski
Life of this patent
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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.
Claims32
A composition for testing a fuel sample for microbial contamination comprising;
- (a) a quantity of hydrocarbon fuel;
- (b) a microbial contamination wherein the microbial contamination further comprises nucleic acid in the form of both DNA, RNA or a combination thereof;
- and
- (c) 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.
A testing chamber for conducting a test of a hydrocarbon fuel for microbial contamination,
- wherein the chamber comprises a sample of a hydrocarbon fuel that is contaminated by at least one microbe;
- wherein the microbial contamination comprises nucleic acid in the form of DNA, RNA or a combination of both;
- wherein the sample is mixed with an analyzing solution;
- wherein the analyzing solution comprises of at least five (5) primer pairs for amplification of at least one target locus;
- and
- wherein at least one of the primer pair binds to the nucleic acid of the microbial contamination.
A method for identifying the microbial contamination of a fuel sample, said contamination comprising nucleic acid in the form of DNA, RNA or a combination thereof, said method comprising the following steps:
- (a) placing said sample in a reaction chamber;
- (b) adding an analyzing solution to said reaction chamber, said solution comprising at least six (6) primer pairs for amplification of at least one target locus, and wherein at least one of the primer pairs binds to the nucleic acid of the microbial contamination;
- (c) performing multiplex PCR amplification;
- (d) electrophoretically separating the resulting amplicons;
- (e) using the fluorescent labels on each amplicon to determine its size;
- (f) correlating the sizes of the amplicons to identify the microorganisms in the fuel.
A method for determining the effectiveness of a biocide treatment, comprising the steps of:
- (a) placing said sample that has been treated with a biocide into a reaction chamber;
- (b) adding an analyzing solution to said reaction chamber, said solution comprising 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;
- wherein at least five or more different types of dyes are used in the solution;
- wherein each type of dye has a unique peak emission wavelength;
- and wherein at least one of the primer pairs binds to the RNA of the microbial contamination;
- of the microbial contamination;
- (c) performing reverse transcription PCR amplification to generate at least one amplicon;
- (d) determining the fragment size of the at least one amplicon;
- (e) correlating the at least one amplicon to the effectiveness of the biocide treatment.
A method for determining the effectiveness of a biocide treatment, comprising the steps of:
- (a) placing said sample that has been treated with a biocide into a reaction chamber;
- (b) combine the sample with a solution containing a DNA-degrading enzyme;
- (c) separating intact microbial cells from the solution of step (b);
- (d) lysing the microbial cells of (c);
- (e) adding an analyzing solution to said reaction chamber, said solution comprising 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;
- wherein at least five or more different types of dyes are used in the solution;
- wherein each type of dye has a unique peak emission wavelength;
- and wherein at least one of the primer pairs binds to the RNA of the microbial contamination;
- (f) performing amplification to generate at least one amplicon,
- (g) determining the fragment size of the at least one amplicon;
- (h) correlating the at least one amplicon to the effectiveness of the biocide treatment.
A composition for testing a fuel for microbial contamination comprising;
- (a) a quantity of hydrocarbon fuel;
- (b) a microbial contamination wherein the microbial contamination further comprises nucleic acid in the form of both DNA and RNA;
- and
- (c) an analyzing solution;
- wherein the analyzing solution comprises of at least six (6) primer pairs for amplification of target loci selected from the loci of any of the tables, FIG. 13, 19, 20, 21, 25, 29-31 or 36;
- wherein at least one primer of each pair of primers is labeled with a fluorescent dye, wherein at least five or more different types of dyes are used in the solution, each type of dye having a unique peak emission wavelength;
- and
- wherein at least one of the primer pair binds to the nucleic acid of the microbial contamination.
A composition for testing a fuel for microbial contamination comprising;
- (a) a quantity of hydrocarbon fuel;
- (b) a microbial contamination wherein the microbial contamination further comprises nucleic acid in the form of both DNA and RNA;
- and
- (c) an analyzing solution;
- wherein the analyzing solution comprises of at least six (6) primer pairs for amplification of target loci selected from the loci of any of the tables of FIG. 13, 19, 20, 21, 25, 29-31 or 36;
- wherein at least one primer of each pair of primers is labeled with a fluorescent dye, wherein at least five or more different types of dyes are used in the solution, each type of dye having a unique peak emission wavelength;
- wherein at least one of the primer pair binds to the nucleic acid of the microbial contamination.
Dependencies are inferred from the claim text.