COMPOSITIONS FOR USE IN IDENTIFICATION OF MEMBERS OF THE BACTERIAL GENUS MYCOPLASMA

• US 20110189687A1
• Filed: 09/30/2009
• Published: 08/04/2011
• Est. Priority Date: 10/02/2008
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Fernandez, Javier P., Sampath, Rangarajan, Li, Feng, Hall, Thomas A., Blyn, Lawrence B.
• Application Number:
  US13/122,359
• Publication Number:
  US 20110189687A1
• Time in Patent Office: Days
• US Class Current: 435/6.15
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention relates generally to identification of members of the bacterial class Mollicutes or the bacterial genus Mycoplasma 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 bacterium 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 bacterium.

2. The primer pair of claim 1 wherein said bacterium is a member of the class Mollicutes, the genus Mycoplasma or the genus Acholeplasma.

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;

3;

6, 1;

9, 11;

12, 4;

14, 13;

16, 18;

7, 15;

2, and 20;

5.

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 bacterium is selected from the group consisting of Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma bovis, Mycoplasma arginini, Mycoplasma orale, Mycoplasma laidlawi, Mycoplasma hominis, Acholeplasma hyorhinis, Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma arthritidis, Mycoplasma mycoides, Afipia felis, Acholeplasma laidlawii.

26. A method for identifying a member of the bacterial class Mollicutes or the bacterial genus Mycoplasma 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 bacteria; and

d) identifying a match between said molecular mass and at least one of saiol database-stored molecular masses of amplification products, thereby identifying said a member of the bacterial genus Mycoplasma.

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;

3;

6, 1;

9, 1;

12, 4;

14, 13;

16, 18;

7, 15;

2 and 20;

5.

28. The method of claim 27 wherein said nucleic acid comprises at least a portion of a gene encoding tufB or rpoB.

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

30. A method for identifying a member of the bacterial class Mollicutes or the bacterial genus Alveoplasma irr a sample, said method comprising:

(a) obtaining an amplification product by amplifying nucleic acid of a bacterium 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 bacteria; and

(e) identifying a match between said base composition and at least one of said database-stored base compositions of amplification products, thereby identifying said member of the bacterial class Mollicutes or the bacterial genus Mycoplasma.

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:

3;

6, 1;

9, 11;

12, 4;

14, 13;

16, 18;

7, 15;

2, and 20;

5.

32. The method of claim 31 wherein said nucleic acid comprises at least a portion of a gene encoding tufB or rpoB.

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

14. An isolated amplification product for identification of a member of the bacterial class Mollicutes or the bacterial genus Mycoplasma, 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 bacteria 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 bacteria, said base composition of said intervening region providing a means for identifying said member of the bacterial genus Mycoplasma; 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;

3;

6, 1;

9, 11;

12, 4;

14, 13;

16, 18;

7, 15;

2, and 20;

5.

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 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-propynyhiracil 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.

39. A system, comprising:

(a) amass 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 members of the bacterial class Mollicutes or the bacterial genus Mycoplasma; 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 member of the bacterial genus Mycoplasma.

40. The system of claim 39 wherein said database of known molecular masses and/or known base compositions of amplification products of mernbers of the bacterial genus Mycoplasma 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 NOs;

3;

6, 1;

9, 111;

12, 4;

14, 113;

16, 18;

7, 15;

2, 20;

5, 8;

10 and 19;

17.

34. A kit comprising one or more purified primer pairs for identifying a. member of bacterial class Mollicutes or the bacterial genus Mycoplasma 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;

3;

6, 1;

9, 11;

12,4;

14, 13;

16, 18;

7, 15;

2, and 20;

5.

35. The kit of claim 34 further comprising one or more primers pair targeted to bacterial ribosomal RNA.

36. The kit of claim 35 wherein said one or more primer pairs targeted to bacterial ribosomal RNA comprise primer pairs having 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;

8;

10 and 19;

17.

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

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