Methods for concurrent identification and quantification of an unknown bioagent
First Claim
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1. A system, comprising:
- a) a nucleic acid amplification component, comprising a pair of primers designed to produce a bioagent identifying amplicon from a bioagent nucleic acid sequence acid under amplification conditions, a known quantity of a calibration polynucleotide comprising a calibration sequence designed to produce a calibration amplicon as a result of amplification with said primers under said amplification conditions wherein the 5′ and
3′
ends of said amplicons are the sequences of said pair of primers or complements thereof, and an amplification reaction vessel wherein said bioagent identifying amplicon and said calibration amplicon are concurrently amplified;
b) a molecular mass determination component; and
c) a molecular mass identification component comprising at least one computer comprising a processor and software, and a database of molecular masses of bioagent identifying amplicons from a plurality known bioagents wherein a match between the molecular mass of said bioagent identifying amplicon and the molecular mass of a bioagent identifying amplicon from a known bioagent in said database of molecular masses identifies said bioagent, a molecular mass of said calibration amplicon identifies said calibration amplicon, and molecular mass abundance data of said bioagent identifying ampicon and molecular mass abundance data of said calibration amplicon indicates the quantity of said bioagent.
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Abstract
The present invention provides methods for the quantification of an unknown bioagent in a sample by amplification of nucleic acid of the bioagent, and concurrent amplification of a known quantity of a calibration polynucleotide from which are obtained a bioagent identifying amplicon and a calibration amplicon. Upon molecular mass analysis, mass and abundance data are obtained. The identity of the bioagent is then determined from the molecular mass of the bioagent identifying amplicon and the quantity of the identified bioagent in the sample is determined from the abundance data of the bioagent identifying amplicon and the abundance data of the calibration amplicon.
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Citations
20 Claims
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1. A system, comprising:
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a) a nucleic acid amplification component, comprising a pair of primers designed to produce a bioagent identifying amplicon from a bioagent nucleic acid sequence acid under amplification conditions, a known quantity of a calibration polynucleotide comprising a calibration sequence designed to produce a calibration amplicon as a result of amplification with said primers under said amplification conditions wherein the 5′ and
3′
ends of said amplicons are the sequences of said pair of primers or complements thereof, and an amplification reaction vessel wherein said bioagent identifying amplicon and said calibration amplicon are concurrently amplified;b) a molecular mass determination component; and c) a molecular mass identification component comprising at least one computer comprising a processor and software, and a database of molecular masses of bioagent identifying amplicons from a plurality known bioagents wherein a match between the molecular mass of said bioagent identifying amplicon and the molecular mass of a bioagent identifying amplicon from a known bioagent in said database of molecular masses identifies said bioagent, a molecular mass of said calibration amplicon identifies said calibration amplicon, and molecular mass abundance data of said bioagent identifying ampicon and molecular mass abundance data of said calibration amplicon indicates the quantity of said bioagent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A system, comprising:
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a) a nucleic acid amplification component, comprising a pair of primers designed to produce a bioagent identifying amplicon from a bioagent nucleic acid sequence acid under amplification conditions, a known quantity of a calibration polynucleotide comprising a calibration sequence designed to produce a calibration amplicon as a result of amplification with said primers under said amplification conditions wherein the 5′ and
3′
ends of said amplicons are the sequences of said pair of primers or complements thereof, and an amplification reaction vessel wherein said bioagent identifying amplicon and said calibration amplicon are concurrently amplified;b) a base composition determination component comprising a component that measures the molecular masses of said bioagent identifying amplicon and said calibration amplicon, and a computer comprising a processor and software that calculates the base compositions of said molecular masses; and c) a base composition identification component comprising at least one computer comprising a processor and software, and a database of base compositions of bioagent identifying amplions from a plurality of known bioagents wherein a match between the base composition of said bioagent identifying amplicon and the base composition of a bioagent identifying amplicon from a known bioagent in said database of base compositions identifies said bioagent, a base composition of said calibration amplicon identifies said calibration amplicon, and molecular mass abundance data of said bioagent identifying ampicon and molecular mass abundance data of said calibration amplicon indicates the quantity of said bioagent. - View Dependent Claims (15, 16, 17, 18, 19, 20)
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Specification