COMPOSITIONS FOR USE IN IDENTIFICATION OF CLOSTRIDIUM DIFFICILE

• US 20110183344A1
• Filed: 09/30/2009
• Published: 07/28/2011
• Est. Priority Date: 10/03/2008
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Sampath, Rangarajan, Li, Feng, Hall, Thomas A., Blyn, Lawrence B., Ecker, David J., Lovari, Robert J.
• Application Number:
  US13/122,364
• Publication Number:
  US 20110183344A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

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

Claims

1. A purified oligonucleotide primer pair for identifying a strain of Clostridium difficile in a sample, said primer pair comprising a forward primer and a reverse primer, each configured to hybridize to nucleic acid of two or more different species or strains of bacteria 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 two or more different species or strains of bacteria, said base composition of said intervening region providing a means for identifying said a strain of Clostridium difficile.

2. The primer pair of claim 1 wherein said strain of Clostridium difficile comprises a subspecies characteristic selected from the group consisting of:

binary toxin cdtA, and binary toxin cdtB, toxin A, toxin B, an 18-nucleobase 18 nucleobase deletion within the tcdC gene, and a single nucleobase deletion within the tcdC gene.

3. The primer pair of claim 2 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;

18;

19, 8;

25, 21;

2, 17;

23, 14;

10, 24;

7, 22;

5, 3;

11, 13;

9, 12;

20, 15;

6, 16;

6 and 1;

4.

4. The primer pair of claim 3 wherein said forward primer and said reverse primer are about 14 to about 40 nucleobases in length.

5. The primer pair of claim 3, wherein said forward primer or said reverse primer or both further comprise a non-templated thymidine residue on the 5′

-end.

6. The primer pair of claim 3, wherein said forward primer or said reverse primer or both further comprise at least one molecular mass modifying tag.

7. The primer pair of claim 3, wherein said forward primer or said reverse primer or both further comprise at least one modified nucleobase.

8. The primer pair of claim 7, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

9. The primer pair of claim 7, wherein said modified nucleobase is a mass-modified nucleobase.

10. The primer pair of claim 9, wherein said mass-modified nucleobase is 5-iodo-cytosine.

11. The primer pair of claim 7, wherein said modified nucleobase is a universal nucleobase.

12. The primer pair of claim 11, wherein said universal nucleobase is inosine.

13. The primer pair of claim 1 wherein said strain of Clostridium difficile is selected from the group consisting of Clostridium difficile Nap1, Clostridium difficile Nap1a, Clostridium difficile IX, Clostridium difficile VII and Clostridium difficile VIII.

26. A method for identifying a strain of Clostridium difficile in a sample said method comprising:

(a) obtaining an amplification product by amplifying nucleic acid of a bacterium in said sample using the primer pair of claim 1;

(b) measuring the molecular mass of one or both strands of said amplification product;

(c) comparing said molecular mass to a plurality of database-stored molecular masses of strands of amplification products of known strains of Clostridium difficile; and

d) identifying a match between said molecular mass and at least one of said database-stored molecular masses of amplification products, thereby identifying said strain of Clostridium difficile.

27. The method of claim 26 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;

18;

19, 8;

25, 21;

2, 17;

23, 14;

10, 24;

7, 22;

5, 3;

11, 13;

9, 12;

20, 15;

6, 16;

6 and 1;

4.

28. The method of claim 27 wherein said nucleic acid comprises a subspecies characteristic selected from the group consisting of:

binary toxin cdtA, and binary toxin cdtB, toxin A, toxin B, an 18-nucleobase 18 nucleobase deletion within the tcdC gene, and a single nucleobase deletion within the tcdC gene.

29. The method of claim 26 wherein said molecular mass is determined by mass spectrometry.

30. A method for identifying a strain of Clostridium difficile in a sample, said method comprising:

(a) obtaining an amplification product by amplifying nucleic acid of a strain of Clostridium difficile in said sample using the purified primer pair of claim 1;

(b) measuring the molecular mass of one or both strands of said amplification product;

(c) determining the base composition of said amplification product from said molecular mass;

(d) comparing said base composition to a plurality of database-stored base compositions of strands of amplification products of known strains of Clostridium difficile; and

(e) identifying a match between said base composition and at least one of said database-stored molecular masses of amplification products, thereby identifying said strain of Clostridium difficile.

31. The method of claim 30 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;

18;

19, 8;

25, 21;

2, 17;

23, 14;

10, 24;

7, 22;

5, 3;

11, 13;

9, 12;

20, 15;

6, 16;

6 and 1;

4.

32. The method of claim 31 wherein said nucleic acid comprises a subspecies characteristic selected from the group consisting of:

binary toxin cdtA, and binary toxin cdtB, toxin A, toxin B, an 18-nucleobase 18 nucleobase deletion within the tcdC gene, and a single nucleobase deletion within the tcdC gene.

33. The method of claim 30 wherein said molecular mass is determined by mass spectrometry.

14. An isolated amplification product for identification of strain of Clostridium difficile, said amplification product produced by a process comprising:

a) amplifying nucleic acid of a bacterium 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 two or more different strains of Clostridium difficile 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 two or more different strains of Clostridium difficile, said base composition of said intervening region providing a means for identifying said strain of Clostridium difficile; and

b) isolating said amplification product from said reaction mixture.

15. The amplification product of claim 14 wherein said isolating step is performed using an anion exchange resin linked to a magnetic bead.

16. The amplification product of claim 14 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;

18;

19, 8;

25, 21;

2, 17;

23, 14;

10, 24;

7, 22;

5, 3;

11, 13;

9, 12;

20, 15;

6, 16;

6 and 1;

4.

17. The amplification product of claim 16 wherein said forward primer and said reverse primer are about 14 to about 40 nucleobases in length.

18. The amplification product of claim 16, wherein said forward primer or said reverse primer or both further comprise a non-templated thymidine residue on the 5′

-end.

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

20. The amplification product of claim 16, wherein said forward primer or said reverse primer or both further comprise at least one modified nucleobase.

21. The amplification product of claim 20, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

22. The amplification product of claim 20, wherein said modified nucleobase is a mass-modified nucleobase.

23. The amplification product of claim 22, wherein said mass-modified nucleobase is 5-iodo-cytosine.

24. The amplification product of claim 22, wherein said modified nucleobase is a universal nucleobase.

25. The amplification product of claim 24, wherein said universal nucleobase is inosine.

37. A system, comprising:

(a) a mass spectrometer configured to detect one or more molecular masses of an amplification product of claim 14;

(b) a database of known molecular masses and/or known base compositions of amplification products of strains of Clostridium difficile; 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 strain of Clostridium difficile.

34. A kit comprising one or more purified primer pairs for identifying a strain of Clostridium difficile in a sample, each member of said one or more primer pairs having at least 70% sequence identity with a corresponding member of one or more primer pairs selected from the group consisting of:

SEQ ID NOs;

18;

19, 8;

25, 21;

2, 17;

23, 14;

10, 24;

7, 22;

5, 3;

11, 13;

9, 12;

20, 15;

6, 16;

6 and 1;

4.

35. The kit of claim 34 further comprising deoxynucleotide triphosphates.

36. The kit of claim 34 wherein one or more of said deoxynucleotide triphosphates is 13C-enriched.

38. The system of claim 47 wherein said database of known molecular masses and/or known base compositions of amplification products of strains of Clostridium difficile 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;

18;

19, 8;

25, 21;

2, 17;

23, 14;

10, 24;

7, 22;

5, 3;

11, 13;

9, 12;

20, 15;

6, 16;

6 and 1;

4.