Methods for rapid identification of pathogens in humans and animals

• US 9,725,771 B2
• Filed: 08/29/2014
• Issued: 08/08/2017
• Est. Priority Date: 12/06/2002
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Blyn, Lawrence B., Ranken, Raymond, Hofstadler, Steven A., Hall, Thomas A., Ecker, David J., Griffey, Richard H., Sampath, Rangarajan, McNeil, John, Crooke, Stanley T.
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/473,603
• Publication Number:
  US 20150056620A1
• Time in Patent Office: 1,075 Days
• Field of Search: None
• 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 of identifying a plurality of etiologic agents of disease in an individual comprising the steps of:

amplifying at least one nucleic acid molecule obtained from a biological sample from the individual with a plurality of primers that hybridize to sequence regions of said nucleic acid that are conserved among different etiologic agents of disease, wherein said conserved sequence regions flank a variable sequence region to obtain a plurality of amplification products, wherein said variable sequence region is within a gene; and

determining the molecular masses of the plurality of amplification products, wherein the molecular masses identify the plurality of etiologic agents and wherein the primers distinguish an etiologic agent of disease at the division level wherein said primers hybridize to said sequence regions that are conserved at the division level, distinguish an etiologic agent of disease at the species level wherein said primers hybridize to sequence regions that are conserved at the species level, or a combination thereof.

2. A method of claim 1 wherein identification of at least one of the plurality of etiologic agents is accomplished at the genus or species level, and the primers distinguish an etiologic agent of disease at the division level wherein said primers hybridize to said sequence regions that are conserved at the division level, distinguish an etiologic agent of disease at the species level wherein said primers hybridize to sequence regions that are conserved at the species level, or a combination thereof.

3. A method of claim 1 wherein a subspecies characteristic of at least one of the plurality of etiologic agents is obtained using primers that distinguish an etiologic agent of disease at the subspecies level wherein said primers hybridize to sequence regions that are conserved at the subspecies level.

4. A method of claim 3 wherein the subspecies characteristic is serotype, strain type, sub-strain type, sub-species type, emm-type, presence of a bioengineered gene, presence of a toxin gene, presence of an antibiotic resistance gene, presence of a pathogenicity island, or presence of a virulence factor.

5. A method of claim 1 wherein the molecular mass is determined by mass spectrometry.

6. A method of claim 5 wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, or triple quadrupole.

7. A method of claim 1 wherein the molecular masses are used to determine the base compositions of the amplification products and wherein the base compositions identify a plurality of etiologic agents of disease.

19. A method of claim 1 wherein said variable sequence regions is within a coding region of a gene.

20. A method of claim 1 wherein said variable sequence region is within a regulatory region of a gene.

8. A method of performing epidemic surveillance comprising the steps of:

amplifying at least one nucleic acid molecule obtained from a plurality of biological samples obtained from a plurality of geographic locations with at least one pair of primers that hybridize to sequence regions of said nucleic acid that are conserved among different pathogens, wherein said conserved sequence regions flank a variable sequence region, wherein said variable sequence region is within a gene, wherein said primers distinguish a pathogen at the division level wherein said primers hybridize to said sequence regions that are conserved at the division level, distinguish a pathogen at the species level wherein said primers hybridize to sequence regions that are conserved at the species level, or a combination thereof to obtain at least one amplification product; and

determining the molecular mass of the at least one amplification product, wherein the molecular mass identifies the pathogen in the biological sample, and wherein identification of a pathogen in a sample from a particular geographic location indicates the spread of the pathogen to the particular geographic location.

9. A method of claim 8 wherein the pathogen is a bacterium, a virus, a protozoan, a parasite, a mold, or a fungus.

10. A method of claim 8 wherein the biological sample is blood, mucus, hair, urine, breath, saliva, sputum, stool, nail, or tissue biopsy.

11. A method of claim 8 wherein the biological sample is obtained from an animal.

12. A method of claim 11 wherein the animal is a human.

13. A method of claim 8 wherein identification of the pathogen is accomplished at the genus or species level, and the primers distinguish a pathogen at the division level wherein said primers hybridize to said sequence regions that are conserved at the division level, or distinguish a pathogen at the species level wherein said primers hybridize to sequence regions that are conserved at the species level.

14. A method of claim 8 wherein a subspecies characteristic about the pathogen is obtained using primers that distinguish a pathogen at the subspecies level wherein said primers hybridize to sequence regions that are conserved at the subspecies level.

15. A method of claim 14 wherein the subspecies characteristic is serotype, strain type, sub-strain type, sub-species type, emm-type, presence of a bioengineered gene, presence of a toxin gene, presence of an antibiotic resistance gene, presence of a pathogenicity island, or presence of a virulence factor.

16. A method of claim 8 wherein the molecular mass is determined by mass spectrometry.

17. A method of claim 16 wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, or triple quadrupole.

18. A method of claim 8 wherein the primers are targeted to ribosomal RNA or housekeeping genes.

21. A method of claim 8 wherein said variable sequence region is within a coding region of a gene.

22. A method of claim 8 wherein said variable sequence region is within a regulatory region of a gene.