Methods for rapid identification of pathogens in humans and animals
View Patent ↗The present invention provides methods of: identifying pathogens in biological samples from humans and animals, resolving a plurality of etiologic agents present in samples obtained from humans and animals, determining detailed genetic information about such pathogens or etiologic agents, and rapid detection and identification of bioagents from environmental, clinical or other samples.
1. A method of identifying a virus comprising:
contacting nucleic acid from said virus with at least one pair of primers which hybridize to flanking sequences of said nucleic acid, wherein said flanking sequences flank a variable nucleic acid sequence of said virus;
amplifying said variable nucleic acid sequence to produce an amplification product;
determining the base composition of said amplification product by mass spectrometry, wherein said base composition identifies the number of A residues, C residues, T residues, G residues, U residues, analogues thereof and mass tag residues thereof in said amplification product; and
comparing said base composition of said amplification product to calculated or measured base compositions of analogous amplification products of one or more known viruses present in a database comprising 5 or more base compositions with the proviso that sequencing of said amplification product is not used to identify the virus.
2. The method of claim 1 , further comprising repeating said contacting, amplifying, determining and comparing steps using one or more additional pairs of primers.
3. The method of claim 1 , wherein said virus is a biological warfare threat agent.
4. The method of claim 1 , wherein said virus is identified at the sub-species level.
5. The method of claim 1 , wherein said virus is a respiratory pathogen.
6. The method of claim 1 , wherein said virus is a hepatitis C virus.
7. The method of claim 1 , wherein said virus is an immunodeficiency virus.
8. The method of claim 1 , wherein said virus is a member of a viral family selected from the group consisting of Filoviridae, Flaviviridae, Arenaviridae, Bunyaviridae, Adenoviridae, Picornaviridae, Togaviridae, and Cononaviridae.
9. The method of claim 1 , wherein said nucleic acid is a housekeeping gene.
10. The method of claim 9 , wherein said housekeeping gene is a polymerase, a virion component, a helicase, a protease, a methyltransferase, or an accessory protein.
11. The method of claim 10 , wherein said polymerase is RNA-dependent RNA polymerase, DNA-dependent DNA polymerase or DNA-dependent RNA polymerase.
12. The method of claim 1 , wherein said amplifying step comprises the polymerase chain reaction.
13. The method of claim 1 , wherein the sequences to which the primers hybridize are separated by between about 60-100 nucleotides.
14. The method of claim 1 , wherein said virus is identified at the species level.
15. The method of claim 1 , wherein said pair of primers comprises at least one nucleotide analog.
16. The method of claim 15 , wherein said nucleotide analog is inosine, uridine, 2,6-diaminopurine, propyne C, or propyne T.
17. The method of claim 1 , wherein a molecular mass-modifying tag is incorporated into said amplification product to limit the number of possible base compositions consistent with the mass of said amplification product.