Methods for rapid identification of pathogens in humans and animals

• US 9,416,424 B2
• Filed: 06/30/2011
• Issued: 08/16/2016
• Est. Priority Date: 03/02/2001
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Ecker, David J., Griffey, Richard H., Sampath, Rangarajan, Hofstadler, Steven A., McNeil, John, Crooke, Stanley T., Blyn, Lawrence, Ranken, Ray, Hall, Thomas A.
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US13/174,254
• Publication Number:
  US 20150284778A1
• Time in Patent Office: 1,874 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 23/00 Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

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.

Claims

1. A method for identifying two or more bioagents, comprising:

a) contacting a sample suspected of containing two or more bioagents with a plurality of primer pairs to simultaneously generate two or more amplicons no more than about 30-250 nucleotides in length, wherein each of said primer pairs hybridize to conserved nucleic acid gene sequences flanking a variable sequence;

wherein the primers of a first primer pair of said plurality of primer pairs hybridize to nucleic acid gene sequences that are conserved within a first genus and flank a variable sequence that varies between species within a first genus and generate an amplicon from a first bioagent but not a second bioagent, and the primers of a second primer pair of said plurality of primer pairs hybridize to nucleic acid gene sequences that are conserved within a second genus and flank a variable sequence that varies between species within a second genus and generate an amplicon from said second bioagent but not said first bioagent, wherein said first and second bioagents differ in genus;

b) simultaneously determining the mass, base composition, or sequence of said two or more amplicons; and

c) simultaneously identifying two or more bioagents in said sample by comparing the determined masses, base compositions, or sequences to a database comprising predetermined masses, base compositions, or sequences from a plurality of known organisms.

2. The method of claim 1, wherein said first bioagent and said second bioagent differ in family.

3. The method of claim 1, wherein said first bioagent and said second bioagent differ in order.

4. The method of claim 1, wherein said first bioagent and said second bioagent differ in class.

5. The method of claim 1, wherein said first bioagent is a bacteria and second bioagent is a virus.

6. The method of claim 1, wherein said one or more amplicons are no more than 50 nucleotides in length.

7. The method of claim 1, wherein two or more amplicons are generated from different regions of nucleic acid from each of said first and second bioagents.

8. The method of claim 1, wherein said determining step is conducted without using radioactive of fluorescent labels.

9. The method of claim 1, wherein said amplicons are isolated in the solid phase prior to or during said determining step.

10. The method of claim 9, wherein said amplicons are isolated in wells of a multi-well plate.

11. A method for identifying two or more bioagents, comprising:

a) contacting a sample suspected of containing two or more bioagents with two or more primer pairs from said two or more bioagents, wherein each of said primer pairs hybridizes to conserved gene sequences flanking a variable sequence, wherein the primers of a first primer pair of said two or more primer pairs hybridize to gene sequences that are conserved within a first genus and flank a variable sequence that varies between species within a first genus, and the primers of a second primer pair of said two or more primer pairs hybridize to gene sequences that are conserved within a second genus and flank a variable sequence that varies between species within a second genus to simultaneously generate two or more amplicons, said amplicons no more than about 30-250 nucleotides in length;

b) simultaneously determining the mass, base composition, or sequence of said amplicons; and

c) simultaneously identifying said two or more bioagents by comparing two or more determined masses, base compositions, or sequences to a database comprising predetermined masses, base compositions, or sequences of known organisms.

12. The method of claim 11, wherein said one or more amplicons are no more than 50 nucleotides in length.

13. The method of claim 11, wherein said determining step is conducted without using radioactive of fluorescent labels.

14. The method of claim 11, wherein said amplicons are isolated in the solid phase prior to or during said determining step.

15. The method of claim 14, wherein said amplicons are isolated in wells of a multi-well plate.

16. A method for identifying two or more bioagents, comprising:

a) contacting a sample suspected of containing two or more bioagents with a plurality of primer pairs to simultaneously generate one or more amplicons no more than about 30-1000 nucleotides in length, wherein each of said primer pairs hybridize to conserved nucleic acid gene sequences flanking a variable sequence wherein the primers of a first primer pair of said plurality of primer pairs hybridize to nucleic acid gene sequences that are conserved within a first genus and flank a variable sequence that varies between species within a first genus, and the primers of a second primer pair of said plurality of primer pairs hybridize to nucleic acid gene sequences that are conserved within a second genus and flank a variable sequence that varies between species within a second genus;

b) simultaneously determining the mass, base composition, or sequence of said two or more amplicons, wherein said amplicons are isolated in the solid phase prior to or during said determining step; and

c) simultaneously identifying said two or more bioagents by comparing the determined masses, base compositions, or sequences to a database comprising predetermined masses, base compositions, or sequences from a plurality of known organisms.

17. The method of claim 16, wherein said determining step is conducted without using radioactive or fluorescent labels.

18. The method of claim 16, wherein said amplicons are no more than about 30-250 nucleotides in length.

19. The method of claim 16, wherein said amplicons are no more than 50 nucleotides in length.