Methods for identification of sepsis-causing bacteria

• US 8,097,416 B2
• Filed: 05/25/2007
• Issued: 01/17/2012
• Est. Priority Date: 09/11/2003
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hall, Thomas A., Sampath, Rangarajan, Harpin, Vanessa, Hofstadler, Steven A., Jiang, Yun
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US11/754,174
• Publication Number:
  US 20080233570A1
• Time in Patent Office: 1,698 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• 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
• 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: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides compositions, kits and methods for rapid identification and quantification of sepsis-causing bacteria by molecular mass and base composition analysis.

Claims

1. A method for identifying a sepsis-causing bacterium in a sample, comprising:

a) amplifying a nucleic acid from said sample using an oligonucleotide primer pair comprising a forward primer and a reverse primer, said forward primer comprising 21-35 linked nucleotides and comprising the sequence of SEQ ID NO;

309, and said reverse primer comprising 24-35 linked nucleotides and comprising the sequence of SEQ ID NO;

1458; and

b) determining the molecular mass of said amplification product by mass spectrometry.

2. The method of claim 1, further comprising comparing said molecular mass to a database comprising a plurality of molecular masses of bioagent identifying amplicons, wherein a match between said determined molecular mass and a molecular mass in said database identifies said sepsis-causing bacterium in said sample.

3. The method of claim 1, further comprising calculating a base composition of said amplification product using said molecular mass.

4. The method of claim 3, further comprising comparing said calculated base composition to a database comprising a plurality of base compositions of bioagent identifying amplicons, wherein a match between said calculated base composition and a base composition included in said database identifies said sepsis-causing bacterium in said sample.

5. The method of claim 1, further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair wherein the primers of each of said at least one additional primer pair are designed to hybridize to a bacterial gene selected from the group consisting of 16S rRNA, 23S rRNA, tufB, rpoB, valS, rplB, and gyrB.

6. The method of claim 1, wherein said molecular mass identifies the presence of said sepsis-causing bacterium in said sample.

7. The method of claim 6, further comprising determining either sensitivity or resistance of said sepsis-causing bacterium in said sample to one or more antibiotics.

8. The method of claim 1, wherein said molecular mass identifies a sub-species characteristic, strain, or genotype of said sepsis-causing bacterium in said sample.

9. A method for identifying at least one sepsis causing bacteria from a sample comprising the steps of:

a) obtaining a sample;

b) contacting at least one nucleic acid from said sample with at least one purified oligonucleotide primer pair from claim 1;

c) performing an amplification reaction, thereby generating at least one amplification product and;

d) analyzing at least one amplification product from step c to identify at least one sepsis causing bacteria in said sample.

10. The method of claim 9 wherein said analyzing step is selected from the group consisting of mass spectrometry analysis, real time PCR analysis, sequencing analysis, hybridization analysis, hybridization protection assay analysis and mass array analysis.

11. The method of claim 10 wherein said mass spectrometry analysis is ESI TOF mass spectrometry.

12. The method of claim 10 wherein said mass spectrometry analysis comprises generating molecular mass data for said amplification product.

13. The method of claim 12 further comprising calculating a base composition from said generated molecular mass data.

14. The method of claim 12 further comprising comparing said molecular mass data to a plurality of molecular masses in a database, wherein said plurality of molecular masses are indexed to said oligonucleotide primer pairs and to a plurality of known sepsis causing bacteria, and wherein a match between said generated molecular mass data and a member of said plurality of molecular masses identifies at least one sepsis causing bacteria in said sample.

15. The method of claim 13 further comprising comparing said base composition to a plurality of base compositions in a database, wherein said plurality of base compositions are indexed to said primer pairs and to a plurality of known sepsis causing bacteria, and wherein a match between said base composition and a member of said plurality of base compositions identifies at least one sepsis causing bacteria in said sample.

16. The method of claim 14 wherein said at least one sepsis causing bacteria is identified by genus, species, sub-species, serotype or genotype.

17. The method of claim 15 wherein said at least one sepsis causing bacteria is identified by genus, species, sub-species, serotype or genotype.

18. The method of claim 10 wherein said at least one amplification product in step d is substantially purified before analysis.

19. The method of claim 18 wherein said at least one amplification product is substantially purified using a magnetic bead covalently linked with an ion exchange resin.