Compositions For Use In Genotyping Of Klebsiella Pneumoniae

• US 20110065111A1
• Filed: 08/27/2010
• Published: 03/17/2011
• Est. Priority Date: 08/31/2009
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Li, Feng, Blyn, Lawrence B., Sampath, Rangarajan
• Application Number:
  US12/870,306
• Publication Number:
  US 20110065111A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The present invention relates generally to identification and characterization of genotypes of Klebsiella pneumoniae and provides methods, compositions and kits useful for this purpose when combined, for example, with molecular mass or base composition analysis.

Claims

1. A method of identifying or characterizing a genotype of Klebsiella pneumoniae in a sample, said method comprising:

amplifying nucleic acid from said sample with a plurality of primer pairs to produce a plurality of amplification products, each primer pair of said plurality of primer pairs comprising a forward primer and a reverse primer, each configured to hybridize to nucleic acid encoding one or more housekeeping genes of Klebsiella pneumoniae and to produce an amplification product having a length of about 29 to about 200 nucleobases;

measuring the molecular masses of one or both strands of said plurality amplification products;

comparing said molecular masses with database-stored molecular masses of bioagent identifying amplicons of characterized genotypes of Klebsiella pneumoniae defined by said plurality of primer pairs, wherein;

a match of each member of said plurality of molecular masses with individual members of said database-stored molecular masses identifies said genotype of Klebsiella pneumoniae, or

a failure to match one or more of said molecular masses with said individual members of said database-stored molecular masses of bioagent identifying amplicons characterizes a previously unknown genotype of Klebsiella pneumoniae.

2. The method of claim 1 wherein said one or more housekeeping genes are selected from the group consisting of:

glyceraldehyde-3-phosphate dehydrogenase (gapA), translation initiation factor IF-2 (infB), maleate dehydrogenase (mdh), glucosephosphate isomerase (pgi), phosphoglycerate mutase (phoE), RNA polymerase, beta subunit (rpoB) and transport protein tonB (tonB).

3. The method of claim 2 wherein each member of each primer pair of said plurality of primer pairs has at least 70% sequence identity with the corresponding member of one or more primer pairs selected from the group consisting of:

SEQ ID NOs;

5;

11, 12;

15, 7;

29, 9;

30, 13;

19, 21;

22, 10;

31, 4;

24, 14;

20, 26;

27, 18;

25, 1;

33, 3;

28, 6;

17, 2;

23, 8;

32, and 16;

33.

4. The method of claim 1 wherein said molecular masses are determined by mass spectrometry.

5. The method of claim 4 wherein said mass spectrometry is electrospray mass spectrometry.

9. The method of claim 1 wherein said molecular masses are determined by mass spectrometry.

10. The method of claim 9 wherein said mass spectrometry is electrospray mass spectrometry.

6. A method of identifying or characterizing a genotype of Klebsiella pneumoniae in a sample, said method comprising:

amplifying nucleic acid from said sample with a plurality of primer pairs to produce a plurality of amplification products, each primer pair of said plurality of primer pairs comprising a forward primer and a reverse primer, each configured to hybridize to nucleic acid encoding one or more housekeeping genes of Klebsiella pneumoniae and to produce an amplification product having a length of about 29 to about 200 nucleobases;

measuring the molecular masses of one or both strands of said plurality of amplification products;

determining the base compositions one or both strands of said amplification products from said molecular masses;

comparing said base compositions with database-stored base compositions of bioagent identifying amplicons of characterized genotypes of Klebsiella pneumoniae defined by said plurality of primer pairs, wherein;

a match of each member of said plurality of base compositions with individual members of said database-stored base compositions identifies said genotype of Klebsiella pneumoniae, or

a failure to match one or more of said base compositions with said members of said database-stored base compositions of bioagent identifying amplicons characterizes a previously unknown genotype of Klebsiella pneumoniae.

7. The method of claim 6 wherein said one or more housekeeping genes are selected from the group consisting of:

glyceraldehyde-3-phosphate dehydrogenase (gapA), translation initiation factor IF-2 (infB), maleate dehydrogenase (mdh), glucosephosphate isomerase (pgi), phosphoglycerate mutase (phoE), RNA polymerase, beta subunit (rpoB) and transport protein tonB (tonB).

8. The method of claim 7 wherein each member of each primer pair of said plurality of primer pairs has at least 70% sequence identity with the corresponding member of one or more primer pairs selected from the group consisting of:

SEQ ID NOs;

5;

11, 12;

15, 7;

29, 9;

30, 13;

19, 21;

22, 10;

31, 4;

24, 14;

20, 26;

27, 18;

25, 1;

33, 3;

28, 6;

17, 2;

23, 8;

32, and 16;

33.

11. A purified oligonucleotide primer pair for identifying a known genotype of Klebsiella pneumoniae or characterizing a previously unknown genotype of Klebsiella pneumoniae, said primer pair comprising a forward primer and a reverse primer, each configured to hybridize to nucleic acid of a plurality of different genotypes of Klebsiella pneumoniae in a nucleic acid amplification reaction which produces an amplification product between about 29 to about 200 nucleobases in length, said amplification product comprising portions corresponding to a forward primer hybridization region, a reverse primer hybridization region and an intervening region having a base composition which varies among amplification products produced from nucleic acid of said plurality of genotypes of Klebsiella pneumoniae, said base composition of said intervening region providing a means for identifying said previously known genotype of Klebsiella pneumoniae or characterizing said previously unknown genotype of Klebsiella pneumoniae.

12. The primer pair of claim 11 wherein said nucleic acid encodes one or more housekeeping genes selected from the group consisting of:

glyceraldehyde-3-phosphate dehydrogenase (gapA), translation initiation factor IF-2 (infB), maleate dehydrogenase (mdh), glucosephosphate isomerase (pgi), phosphoglycerate mutase (phoE), RNA polymerase, beta subunit (rpoB) and transport protein tonB (tonB).

13. The primer pair of claim 12 wherein each member of said primer pair has at least 70% sequence identity with a corresponding member of a primer pair selected from the group consisting of:

SEQ ID NOs;

5;

11, 12;

15, 7;

29, 9;

30, 13;

19, 21;

22, 10;

31, 4;

24, 14;

20, 26;

27, 18;

25, 1;

33, 3;

28, 6;

17, 2;

23, 8;

32, and 16;

33.

14. The primer pair of claim 13 wherein said forward primer and said reverse primer are about 14 to about 40 nucleobases in length.

15. The primer pair of claim 13, wherein said forward primer or said reverse primer or both further comprise a non-templated thymidine residue on the 5′

-end.

16. The primer pair of claim 13, wherein said forward primer or said reverse primer or both further comprise at least one molecular mass modifying tag.

17. The primer pair of claim 13, wherein said forward primer or said reverse primer or both further comprise at least one modified nucleobase.

18. The primer pair of claim 17, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

19. The primer pair of claim 17, wherein said modified nucleobase is a mass-modified nucleobase.

20. The primer pair of claim 17, wherein said mass-modified nucleobase is 5-iodo-cytosine.

21. The primer pair of claim 17, wherein said modified nucleobase is a universal nucleobase.

22. The primer pair of claim 21, wherein said universal nucleobase is inosine.

26. A kit for identifying a known genotype of Klebsiella pneumoniae or characterizing a previously unknown genotype of Klebsiella pneumoniae, said kit comprising a plurality of primer pairs according to claim 11.

27. The kit of claim 26 wherein said nucleic acid encodes one or more housekeeping genes selected from the group consisting of:

glyceraldehyde-3-phosphate dehydrogenase (gapA), translation initiation factor IF-2 (infB), maleate dehydrogenase (mdh), glucosephosphate isomerase (pgi), phosphoglycerate mutase (phoE), RNA polymerase, beta subunit (rpoB) and transport protein tonB (tonB).

28. The kit of claim 27 wherein said plurality of primer pairs comprises a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

5 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

11.

29. The kit of claim 28 further comprising a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

12 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

15.

30. The kit of claim 29 further comprising a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

7 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

29.

31. The kit of claim 30 further comprising a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

9 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

30.

32. The kit of claim 31 further comprising a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

13 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

19.

33. The kit of claim 32 further comprising a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

21 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

22.

34. The kit of claim 33 further comprising a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

10 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

31.

35. The kit of claim 34 further comprising a primer pair with a forward primer having at least 70% sequence identity with SEQ ID NO:

4 and a reverse primer having at least 70% sequence identity with SEQ ID NO;

24.

36. The kit of claim 35 further comprising deoxynucleotide triphosphates.

37. The kit of claim 36 wherein one or more of said deoxynucleotide triphosphates is ¹³C-enriched.

23. An isolated amplification product for use in identification of a known genotype of Klebsiella pneumoniae or characterization of a previously unknown genotype of Klebsiella pneumoniae, said amplification product produced by a process comprising:

a) amplifying nucleic acid of a genotype of Klebsiella pneumoniae in a reaction mixture comprising a primer pair, said primer pair comprising a forward primer and a reverse primer, each configured to hybridize to nucleic acid of a plurality of different genotypes of Klebsiella pneumoniae in a nucleic acid amplification reaction, said amplification product having a length of about 29 to about 200 nucleobases and comprising portions corresponding to a forward primer hybridization region, a reverse primer hybridization region and an intervening region having a base composition which varies among amplification products produced from nucleic acid of said plurality of different genotypes of Klebsiella pneumoniae, said base composition of said intervening region providing a means for identifying said previously known genotype of Klebsiella pneumoniae or characterizing said previously unknown genotype of Klebsiella pneumoniae;

and

b) isolating said amplification product from said reaction mixture.

24. The amplification product of claim 23 wherein step b) is performed using an anion exchange resin linked to a magnetic bead.

25. The amplification product of claim 23 wherein each member of said primer pair has at least 70% sequence identity with a corresponding member of a primer pair selected from the group consisting of:

SEQ ID NOs;

5;

11, 12;

15, 7;

29, 9;

30, 13;

19, 21;

22, 10;

31, 4;

24, 14;

20, 26;

27, 18;

25, 1;

33, 3;

28, 6;

17, 2;

23, 8;

32, and 16;

33.

38. A system, comprising:

(a) a mass spectrometer configured to detect one or more molecular masses of an amplification product of claim 23;

(b) a database of known molecular masses and/or known base compositions of amplification products of known genotypes of Klebsiella pneumoniae;

and

(b) a controller operably connected to said mass spectrometer and to said database, said controller configured to match said molecular masses of said amplification product with a measured or calculated molecular mass of a corresponding amplification product of a known genotype of Klebsiella pneumoniae.

39. The system of claim 38 wherein said database of known molecular masses and/or known base compositions of amplification products of known genotypes of Klebsiella pneumoniae includes amplification products defined by one or more primer pairs wherein each member of said one or more primer pairs has at least 70% sequence identity with a corresponding member of a corresponding primer pair selected from the group consisting of:

SEQ ID NOs;

5;

11, 12;

15, 7;

29, 9;

30, 13;

19, 21;

22, 10;

31, 4;

24, 14;

20, 26;

27, 18;

25, 1;

33, 3;

28, 6;

17, 2;

23, 8;

32, and 16;

33.