PCR ready compositions and methods for detecting and identifying nucleic acid sequences
Inventors
Fischer, Gerald W. • Daum, Luke T.
Assignees
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Publication Number
US-11041215-B2
Publication Date
2021-06-22
Expiration Date
Abstract
The invention is directed to compositions and methods for isolating, detecting, amplifying, and quantitating pathogen-specific nucleic acids in a biological sample, and in particular PCR ready compositions that contain enzyme and are stable or long periods of time. The invention also provides diagnostic kits containing specific amplification primers, and labeled detection probes that specifically bind to the amplification products obtained therefrom. Also disclosed are compositions and methods for the isolation and characterization of nucleic acids that are specific to one or more pathogens, including for example Influenza virus and Mycobacterium tuberculosis, from a wide variety of samples including those of biological, environmental, clinical and/or veterinary origin.
Core Innovation
The invention provides a PCR ready composition that is an aqueous mixture including RNase-free water, a buffer, forward and reverse amplification primers specific for PCR amplification of a target nucleic acid sequence, a plurality of nucleotides sufficient for PCR amplification of the target nucleotide sequence, and a PCR polymerase and/or a reverse transcriptase. The composition further includes a nucleic acid probe consisting of at least a sequence selected from SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO: 33. The primers, the plurality of nucleotides, and the enzyme remain stable in the mixture for at least 5 days at ambient temperature.
In more defined embodiments, the composition includes additional formulation components combined in nuclease-free water, including a chelating agent, an osmolarity agent, albumin, a salt, and a dye, together with the primers, nucleotides, and PCR polymerase and/or reverse transcriptase. The buffer is present at at least 50 mM to about 1 M with a pH of about 6.5 to about 9.0, and the components are formulated with specified chemical options for buffering agents, chelating agents, osmolarity agents, albumin types, salts, and dyes.
The composition is further characterized by stability-related constraints and probe behavior. The selected pH may be constrained relative to the pKa of the buffer at ambient temperature, and the primers, nucleotides, and enzyme can be stable for at least 5 days at ambient temperature and, in some refinements, at least a month. The nucleic acid probe hybridizes to a PCR amplification product of the target nucleic acid, supporting probe-based detection in PCR-ready formulations.
Claims Coverage
The partial content includes three independent claims. Across these claims, the central inventive theme is a PCR-ready nuclease-free aqueous mixture with specified nucleic-acid assay components and stability of primers, nucleotides, and enzyme for at least 5 days at ambient temperature, with defined probe sequence options.
PCR-ready aqueous mixture with RNase-free water, primers, nucleotides, enzyme, and probe sequences
A PCR ready composition comprising an aqueous mixture of RNase-free water; a buffer at a concentration of at least 50 mM; forward and reverse amplification primers specific for PCR amplification of a target nucleic acid sequence; a plurality of nucleotides sufficient for PCR amplification of the target nucleotide sequence; a PCR polymerase enzyme and/or a reverse transcriptase; and a nucleic acid probe that consists of at least a sequence selected from the group of sequences consisting of SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO: 33, wherein the primers, the plurality of nucleotides and the enzyme remain stable in the mixture for at least 5 days at ambient temperature.
PCR-ready aqueous composition with defined buffer, chelating agent, osmolarity agent, albumin, salt, and dye
A PCR-ready composition comprising a buffer present in the composition at a concentration of at least 50 mM to about 1 M and with a pH of from about 6.5 to about 9.0; a chelating agent present in the composition at a concentration of about 0.01 mM to about 1 mM; an osmolarity agent present in the composition at a concentration of about 1 mM to about 1 M; an albumin present in the composition at a concentration of from about 5 ng/ml to about 100 ng/ml; a salt present in the composition at a concentration of from about 50 mM to about 1 M; a dye present in the composition at a concentration of from about 0.01 mM to 50 mM; forward and reverse amplification primers specific for PCR amplification of a target nucleic acid sequence; a plurality of nucleotides sufficient for PCR amplification of the target nucleotide sequence; a PCR polymerase and/or a reverse transcriptase; and a nucleic acid probe consisting of a sequence selected from the group of sequences consisting of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 33, wherein the components are combined in nuclease-free water; and the primers, the plurality of nucleotides and the enzyme remain stable in the mixture for at least 5 days at ambient temperature.
PCR-ready composition including salts, albumin, and non-ionic detergent with glycerol and betaine
A PCR-ready composition comprising a buffer present in the composition at a concentration of at least 50 mM to about 1 M and with a pH of from about 6.5 to about 9.0; a chelating agent present in the composition at a concentration of about 0.01 mM to about 1 mM; forward and reverse amplification primers specific for PCR amplification of a target nucleic acid sequence; a plurality of nucleotides sufficient for PCR amplification of the target nucleotide sequence; at least two salts collectively present in the composition at a concentration of about 50 mM to about 10 M; albumin present in the composition at a concentration of about 5 ng/ml to about 1 mg/ml; a non-ionic detergent, a glycerol and betaine collectively present in the composition at a concentration of about 1 mM to about 1 M; a dye present in the composition at about 0.01 μM to about 1 μM; a PCR polymerase enzyme and/or a reverse transcriptase; and a nucleic acid probe consisting of a sequence selected from the group of sequences consisting of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 33, wherein the components are combined in nuclease-free water; and the primers, the plurality of nucleotides and the enzyme remain stable in the mixture for at least 5 days at ambient temperature.
All independent claims define PCR-ready nuclease-free aqueous compositions with specific assay constituents and require ambient temperature stability of primers, nucleotides, and enzyme for at least 5 days, while dependent refinements further constrain formulation parameters and probe behavior.
Stated Advantages
Primers, plurality of nucleotides, and enzyme remain stable in the mixture for at least 5 days at ambient temperature.
The composition can maintain stability with refinements extending stability for at least a month.
Probe hybridizes to a PCR amplification product of the target nucleic acid.
Documented Applications
Pathogen-specific nucleic-acid detection and quantitation using PCR-ready reagent systems, including target pathogens such as Mycobacterium tuberculosis complex (e.g., IS6110) and Influenza (e.g., H1N1/H5N1).
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