Compositions for use in identification of bacteria

• US 8,288,523 B2
• Filed: 03/07/2007
• Issued: 10/16/2012
• Est. Priority Date: 09/11/2003
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Sampath, Rangarajan, Hall, Thomas A., Ecker, David J., Blyn, Lawrence
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US11/683,254
• Publication Number:
  US 20120122097A1
• Time in Patent Office: 2,050 Days
• Field of Search: None
• US Class Current: 536/24.33
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

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

Claims

1. An oligonucleotide primer pair comprising a forward and a reverse primer, wherein said forward primer comprises SEQ ID NO:

590, and wherein said reverse primer comprises SEQ ID NO;

891.

2. The oligonucleotide primer pair of claim 1 wherein said forward primer is SEQ ID NO:

590.

3. The oligonucleotide primer pair of claim 1 wherein said reverse primer is SEQ ID NO:

891.

4. The oligonucleotide primer pair of claim 1 wherein at least one of said forward primer and said reverse primer comprises at least one modified nucleobase.

5. The oligonucleotide primer pair of claim 4 wherein at least one of said at least one modified nucleobase is a mass modified nucleobase.

6. The oligonucleotide primer pair of claim 5 wherein said mass modified nucleobase is 5-Iodo-C.

7. The composition of claim 5 wherein said mass modified nucleobase comprises a molecular mass modifying tag.

8. The oligonucleotide primer pair of claim 4 wherein at least one of said at least one modified nucleobase is a universal nucleobase.

9. The oligonucleotide primer pair of claim 8 wherein said universal nucleobase is inosine.

10. The oligonucleotide primer pair of claim 1 wherein at least one of said forward primer and said reverse primer comprises a non-templated T residue at its 5′

end.

11. A kit for identifying, determining one or more characteristics of, or detecting a Staphylococcus aureus bioagent comprising the oligonucleotide primer pair of claim 1 and at least one additional primer pair designed to hybridize to a Staphylococcus aureus gene encoding arcC, aroE, gmk, pta, tpi, yqi, or a combination thereof.

12. The kit of claim 11 further comprising at least one other additional primer pair designed to hybridize to a Staphylococcus aureus gene encoding mecA, mecR1, pvluk, or a combination thereof.

13. The kit of claim 11 wherein said at least one additional primer pair comprises SEQ ID NOs:

437;

1232, SEQ ID NOs;

268;

1284, SEQ ID NOs;

418;

1301, SEQ ID NOs;

318;

1300, SEQ ID NOs;

440;

1076, SEQ ID NOs;

219;

1013, or a combination thereof.

14. The kit of claim 11 wherein said oligonucleotide primer pair of claim 1 and said at least one additional primer pair consists of eight oligonucleotide primer pairs wherein at least one of the primers of said at least one additional primer pair has at least 70% sequence identity with at least one of the primers of the primer pairs represented by:

SEQ ID NOs;

437;

1232, SEQ ID NOs;

590;

891, SEQ ID NOs;

474;

869, SEQ ID NOs;

268;

1284, SEQ ID NOs;

418;

1301, SEQ ID NOs;

318;

1300, SEQ ID NOs;

440;

1076, and SEQ ID NOs;

219;

1013.

15. A method for identifying, determining one or more characteristics of, or detecting a Staphylococcus aureus bioagent in a sample comprising:

a) amplifying a nucleic acid from said sample using the oligonucleotide primer pair of claim 1; and

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

16. The method of claim 15 further comprising comparing said determined molecular mass to a plurality of molecular masses of bioagent identifying amplicons, each indexed to said oligonucleotide primer pair and a Staphylococcus aureus bioagent, wherein a match between said determined molecular mass and one of said plurality of molecular masses identifies, determines one or more characteristic of, or detects said Staphylococcus aureus bioagent in said sample.

17. The method of claim 15 further comprising calculating a base composition of said at least one amplification product using said molecular mass.

18. The method of claim 17 further comprising comparing said calculated base composition to a database comprising a plurality of base compositions of bioagent identifying amplicons, each indexed to said oligonucleotide primer pair and a Staphylococcus aureus bioagent, wherein a match between said calculated base composition and a base composition in said database identifies, determines one or more characteristics of, or detects said Staphylococcus aureus bioagent in said sample.

19. The method of claim 15 further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair designed to hybridize to a Staphylococcus aureus gene encoding arcC, aroE, gmk, pta, tpi, yqi or a combination thereof.