Methods for rapid detection and identification of bioagents for environmental testing
OwnerDavid J. Ecker, Richard H. Griffey +3inventors
InventorsDavid J. Ecker, Richard H. Griffey, Rangarajan Sampath, Steven Hofstadler, John McNeil
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Abstract
Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a “base composition signature” (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.
Claims49
A method of identifying an unknown bioagent in an environmental sample comprising:
- a) contacting nucleic acid from said bioagent in said environmental sample with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of the bioagent;
- b) amplifying said variable nucleic acid sequence to produce an amplification product;
- c) determining the molecular mass of said amplification product;
- and
- d) comparing said molecular mass to one or more molecular masses of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein a match identifies said unknown bioagent in said environmental sample.
A method of identifying an unknown bioagent in an environmental sample comprising:
- a) contacting nucleic acid from said bioagent in an environmental sample with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence;
- b) amplifying said variable nucleic acid sequence to produce an amplification product;
- c) determining the base composition of said amplification product;
- and
- d) comparing said base composition to one or more base compositions of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein a match identifies said unknown bioagent in said environmental sample.
A method of determining the absence of a bioagent in an environmental sample comprising:
- a) contacting said environmental sample suspected of containing nucleic acid encoding said bioagent with at least one pair of oligonucleotide primers which are capable of hybridizing to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of said bioagent;
- b) treating said variable nucleic acid sequence under amplification conditions capable of producing an amplification product of said variable nucleic acid sequence;
- c) performing spectroscopy to determine the molecular mass or base composition of all amplification products;
- and
- d) comparing said molecular mass to one or more molecular masses of amplification products or said base composition to one or more base compositions of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein the lack of a match indicates that said bioagent is absent from said environmental sample.
Dependencies are inferred from the claim text.