SECONDARY STRUCTURE DEFINING DATABASE AND METHODS FOR DETERMINING IDENTITY AND GEOGRAPHIC ORIGIN OF AN UNKNOWN BIOAGENT THEREBY

• US 20110238316A1
• Filed: 01/09/2004
• Published: 09/29/2011
• Est. Priority Date: 06/26/2001
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hofstadler, Steven A., Sampath, Rangarajan, Griffey, Richard H., McNeil, John, Ecker, David J., Crooke, Stanley T.
• Granted Patent: US 8,073,627 B2
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

The present invention relates generally to the field of investigational bioinformatics and more particularly to secondary structure defining databases. The present invention further relates to methods for interrogating a database as a source of molecular masses of known bioagents for comparing against the molecular mass of an unknown or selected bioagent to determine either the identity of the selected bioagent, and/or to determine the origin of the selected bioagent. The identification of the bioagent is important for determining a proper course of treatment and/or irradication of the bioagent in such cases as biological warfare. Furthermore, the determination of the geographic origin of a selected bioagent will facilitate the identification of potential criminal identity.

Claims

1-20. -20. (canceled)

21. A method for identifying a pathogen comprising:

a.) providing a system, said system comprising;

i.) an automatic fluidics control system configured to receive two or more amplification products of nucleic acid of said pathogen, said two or more amplification products produced with two or more primer pairs;

ii.) a mass spectrometer configured to directly receive said two or more amplification products from said fluidics control system and configured to provide two or more molecular masses of said two or more amplification products;

iii.) a computer program stored on a computer readable medium configured to receive and convert said two or more molecular masses to two or more base compositions wherein said base compositions identify the number but not the nucleic acid gene sequence order of A residues, C residues, T residues, G residues, U residues, analogues thereof or mass tag residues thereof in said two or more amplification products without sequencing said two or more amplification products;

iv.) a database stored on a computer-readable medium, said database comprising at least nineteen base compositions of amplification products of nucleic acid of at least nineteen known pathogens produced with a plurality of primer pairs wherein said plurality of primer pairs comprises said two or more primer pairs and wherein said two or more base compositions are indexed to the corresponding primer pair and to a pathogen;

v.) a pathogen identification module configured to receive said two or more base compositions from said computer program, and configured to query said received two or more base compositions against said database and configured to determine a match between said two or more base compositions and a member of said at least nineteen base compositions of said database; and

vii.) a display interface configured to produce a pathogen identification report from said match;

b.) obtaining nucleic acid of said pathogen;

c.) amplifying said nucleic acid with said two or more primer pairs to obtain two or more amplification products, wherein at least one of said two or more primer pairs hybridizes to conserved coding regions of a nucleic acid gene sequence wherein said conserved regions flank at least one variable coding region of said nucleic acid gene sequence of said pathogen;

d.) transferring said two or more amplification products to said mass spectrometer using said automated fluidics control system;

e.) measuring two or more molecular masses of said two or more amplification products using said mass spectrometer;

f.) converting said two or more molecular masses to two or more base compositions using said computer program without sequencing said two or more amplification products; and

g.) determining a match between said two or more base compositions and a member of said plurality of base compositions of said database using said pathogen identification module, wherein said member of said at least nineteen base compositions is a known amplification product of a known pathogen produced with said two or more primer pairs, and wherein said match identifies said pathogen.

22. The method of claim 21 wherein said pathogen is a bacterium.

23. The method of claim 21 wherein said pathogen is a virus.

24. The method of claim 21 wherein said pathogen is a fungus.

25. The method of claim 21 wherein said determining step identifies said pathogen at the genus level.

26. The method of claim 21 wherein said determining step identifies said pathogen at the species level.

27. The method of claim 21 wherein said determining step identifies said pathogen at the sub-species level.

28. The method of claim 21 wherein said system further comprises:

vii.) a thermocycler configured to;

provide said two or more amplification products to said fluidics control system.

43. The method of claim 22 wherein said bacterium is a pathogen selected from the group consisting of:

Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.

44. The method of claim 22 wherein said bacterium is a pathogen selected from the group consisting of:

Acinetobacter calcoaceticus, Bacillus anthracis, Bacillus cereus, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella abortus, Campylobacter jejuni, Chlamydia pnuemoniae, Clostridium botulinum, Clostridium difficile, Enterococcus faecalis, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Mycobacterium avium, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Rickettsia prowazekii, Rickettsia rickettsii, Salmonella typhimurium, Shigella dysenteriae, Staphylococcus aureus, Streptomyces, Treponema pallidum, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis.

45. The method of claim 22 wherein said bacterium is a biowarfare agent.

46. The method of claim 45 wherein said biowarfare agent is selected from the group consisting of:

Bacillus anthracis, Yersinia pestis, Franciscella tularensis, Brucella suis, Brucella abortus, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomalleii, Salmonella typhii, Salmonella typhimurium, Rickettsia typhii, Rickettsia rickettsii, Rickettsia prowasekii, Coxiella burnetii, Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, or Vibrio cholerae.

29. A system for identifying a pathogen, said system comprising:

a.) an automatic fluidics control system configured to receive two or more amplification products of nucleic acid of said pathogen, said two or more amplification products produced with two or more primer pairs wherein at least one of said two or more primer pairs hybridizes to conserved coding regions of a nucleic acid gene sequence wherein said conserved regions flank at least one variable coding region of said nucleic acid gene sequence of said pathogen;

b.) a mass spectrometer configured to directly receive said two or more amplification products from said fluidics control system and configured to provide two or more molecular masses of said two or more amplification products;

c.) a computer program stored on a computer readable medium configured to receive and convert said two or more molecular masses to two or more base compositions wherein said base compositions identify the number but not the nucleic acid gene sequence order of A residues, C residues, T residues, G residues, U residues, analogues thereof or mass tag residues thereof in said two or more amplification products without sequencing said two or more amplification products;

d.) a database stored on a computer-readable medium, said database comprising at least nineteen base compositions of amplification products of nucleic acid of at least nineteen known pathogens produced with a plurality of primer pairs wherein said plurality of primer pairs comprises said two or more primer pairs and wherein said two or more base compositions are indexed to the corresponding primer pair and to a pathogen;

e.) a pathogen identification module configured to receive said two or more base compositions from said computer program, and configured to query said received two or more base compositions against said database and configured to determine a match between said two or more base compositions and a member of said at least nineteen base compositions of said database; and

f.) a display interface configured to produce a pathogen identification report from said match.

30. The system of claim 29 further comprising:

g.) a thermocycler configured to;

provide said two or more amplification products to said fluidics control system.

31. The system of claim 30 further comprising:

h.) a reaction vessel wherein said reaction vessel comprises a 96-well or a 384-well microtiter plate.

32. The system of claim 29 wherein said mass spectrometer is an electrospray time-of-flight mass spectrometer.

33. The system of claim 29 wherein said database is a local database.

34. The system of claim 33 wherein said local database is updated by inclusion of additional base compositions of amplification products of additional pathogens from a master database at a remote location.

35. The system of claim 33 wherein said local database is updated by inclusion of additional base compositions of amplification products of additional pathogens from a transferable computer formatted medium.

36. The system of claim 29 wherein said at least nineteen base compositions are base compositions from amplification products of at least one essential gene or at least one nucleic acid encoding ribosomal RNA.

38. The system of claim 29 wherein said mass spectrometer comprises a signal processor configured to analyze mass spectral peaks and provide said two or more molecular masses.

39. The system of claim 29 wherein said automated fluidics control system is configured for high throughput mass spectrometry analysis of a plurality of amplification products at a rate of about one sample per minute.

41. The system of claim 36 wherein said at least one essential gene is selected from the group consisting of genes that express polypeptides involved in translation, replication, recombination, repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, secretion, uptake and combinations thereof.

42. The system of claim 36 wherein said at least one essential gene is selected from the group consisting of genes that encode DNA polymerase, elongation factor TU, heat shock protein, groEL, RNA polymerase, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase, elongation factor G and combinations thereof.

37. (canceled)

40. A system for identifying a pathogen, said system comprising:

a.) an automatic fluidics control system configured to receive two or more amplification products of nucleic acid of said pathogen, said two or more amplification products produced with two or more primer pairs wherein at least one of said two or more primer pairs hybridizes to conserved coding regions of a nucleic acid gene sequence wherein said conserved regions flank at least one variable coding region of said nucleic acid gene sequence of said pathogen;

b.) a mass spectrometer configured to directly receive said two or more amplification products from said fluidics control system and configured to provide two or more molecular masses of said two or more amplification products;

c.) a computer program stored on a computer readable medium configured to receive and convert said two or more molecular masses to two or more base compositions wherein said base compositions identify the number but not the nucleic acid gene sequence order of A residues, C residues, T residues, G residues, U residues, analogues thereof or mass tag residues thereof in said two or more amplification products without sequencing said two or more amplification products;

d.) a database stored on a computer-readable medium, said database comprising at least nineteen base compositions of amplification products of nucleic acid of at least nineteen known pathogens produced with a plurality of primer pairs wherein said plurality of primer pairs comprises said two or more primer pairs and wherein said two or more base compositions are indexed to the corresponding primer pair and to a pathogen; and

,

e.) a pathogen identification module configured to receive said two or more base compositions from said computer program, and configured to query said received two or more base compositions against said database and configured to determine a match between said two or more base compositions and a member of said at least nineteen base compositions of said database.