COMPOSITIONS FOR USE IN IDENTIFICATION OF MEMBERS OF THE BACTERIAL CLASS ALPHAPROTEOBACTER

• US 20110183343A1
• Filed: 09/30/2009
• Published: 07/28/2011
• Est. Priority Date: 10/03/2008
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Sampath, Rangarajan, Li, Feng, Blyn, Lawrence B., Ecker, David J., Lovari, Robert J.
• Application Number:
  US13/122,352
• Publication Number:
  US 20110183343A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The present invention relates generally to identification of members of the bacterial class Alphaproteobacter and provides methods, compositions and kits useful for this purpose when combined, for example, with molecular mass or base composition analysis.

Claims

1. A composition, comprising at least two oligonucleotide primer pairs that are selected from the group consisting of:

at least one primer pair comprising sequences that are configured to generate amplicons comprising a [A₂₈T₃₂C₂₉G₃₃] base composition from a Bartonella elizabethae nucleic acid, a [A₃₀T₃₀C₃₀G₃₂] base composition from a Bartonella doshiae nucleic acid, and a [A₃₁T₃₀C₂₈G₃₃] base composition from a Bartonella quintana nucleic acid;

at least one primer pair comprising sequences that are configured to generate amplicons comprising a [A₂₆T₃₅C₂₉G₃₅] base composition from a Bartonella elizabethae nucleic acid, a [A₂₈T₃₃C₃₀G₃₄] base composition from a Bartonella doshiae nucleic acid, and a [A₂₉T₃₃C₂₈G₃₅] base composition from a Bartonella quintana nucleic acid;

at least one primer pair comprising sequences that are configured to generate amplicons comprising a [A₃₁T₃₁C₁₀G₂₅] base composition from a Bartonella koehlerae nucleic acid, a [A₂₉T₃₃C₁₀G₂₅] base composition from a Bartonella doshiae nucleic acid, and a [A₂₉T₃₃C₁₃G₂₂] base composition from a Bartonella phoceensis nucleic acid;

at least one primer pair comprising sequences that are configured to generate amplicons comprising a [A₂₆T₃₅C₁₈G₃₀] base composition from a Bartonella elizabethae nucleic acid, a [A₂₅T₃₃C₂₀G₃₁] base composition from a Bartonella doshiae nucleic acid, and a [A₂₆T₃₃C₁₈G₃₂] base composition from a Bartonella quintana nucleic acid;

at least one primer pair comprising sequences that are configured to generate amplicons comprising a [A₁₈T₂₃C₁₁G₂₆] base composition from a Bartonella elizabethae nucleic acid, a [A₁₈T₂₂C₂₀G₂₆] base composition from a Bartonella doshiae nucleic acid, and a [A₁₈T₂₂C₁₃G₂₅] base composition from a Bartonella quintana nucleic acid; and

,

at least one primer pair comprising sequences that are configured to generate amplicons comprising a [A₂₅T₃₀C₂₂G₃₅] base composition from a Bartonella elizabethae nucleic acid, a [A₂₆T₃₁C₂₀G₃₅] base composition from a Bartonella doshiae nucleic acid, and a [A₂₅T₃₁C₂₀G₃₆] base composition from a Bartonella quintana nucleic acid.

26. A method for identifying a member of the bacterial class Alphaproteobacter in a sample said method comprising:

(a) obtaining an amplification product by amplifying nucleic acid of a member of the bacterial class Alphaproteobacter 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 members of the bacterial class Alphaproteobacter; 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 a member of the bacterial class Alphaproteobacter.

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;

10;

8, 11;

7, 2;

3, 14;

13, 15;

1, 16;

6, 9;

5, 12;

4, 17;

18, and 19;

20.

28. The method of claim 26 wherein said nucleic acid comprises at least a portion of a gene encoding citrate synthase (gltA), chaperonin GroEL (GroEL), RNA polymerase beta (rpoB) and RNase P.

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 Alphaproteobacter in a sample, said method comprising:

(a) obtaining an amplification product by amplifying nucleic acid of a member of the bacterial class Alphaproteobacter 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 members of the bacterial class Alphaproteobacter; 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 Alphaproteobacter.

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;

10;

8, 11;

7, 2;

3, 14;

13, 15;

1, 16;

6, 9;

5, 12;

4, 17;

18, and 19;

20.

32. The method of claim 30 wherein said nucleic acid comprises at least a portion of a gene encoding citrate synthase (gltA), chaperonin GroEL (GroEL), RNA polymerase beta (rpoB) and RNase P

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

2. A purified oligonucleotide primer pair for identifying a member of the bacterial class Alphaproteobacter, 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 members of the bacterial class Alphaproteobacter 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 members of the bacterial class Alphaproteobacter, said base composition of said intervening region providing a means for identifying said member of the bacterial class Alphaproteobacter.

3. The primer pair of claim 2 wherein said member of the bacterial class Alphaproteobacter is selected from the group consisting of:

Bartonella alsatica, Bartonella arapensis, Bartonella birtlesii, Bartonella bovis, Bartonella broomii, Bartonella chomelii, Bartonella cleveland, Bartonella doshiae, Bartonella elizabethae, Bartonella felis, Bartonella grahamii, Bartonella henselae, Bartonella koehlerae, Bartonella organo, Bartonella phoceensis, Bartonella quintana, Bartonella rhizobi, Bartonella schoenb., Bartonella Sps. A1, Bartonella Sps. A3, Bartonella Sps. A4, Bartonella Sps. A5, Bartonella Sps. B1, Bartonella Sps. B3, Bartonella Sps. B4, Bartonella Sps. C1, Bartonella tamiae, Bartonella vinsonii, Bartonella washoensis, Rickettsiae. conorii, Rickettsiae typhi, Rickettsiae prowazekii, Rickettsiae prowazekii, Rickettsiae rickettsii, Rickettsiae australis, Rickettsiae sibirica, Rickettsiae parkeri and Rickettsiae africae.

4. 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;

10;

8, 11;

7, 2;

3, 14;

13, 15;

1, 16;

6, 9;

5, 12;

4, 17;

18, and 19;

20.

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

6. The primer pair of claim 4, wherein said forward primer or said reverse primer or both further comprise a non-templated thymidine residue on the 5′

-end.

7. The primer pair of claim 4, wherein said forward primer or said reverse primer or both further comprise at least one molecular mass modifying tag.

8. The primer pair of claim 4, wherein said forward primer or said reverse primer or both further comprise at least one modified nucleobase.

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

10. The primer pair of claim 8, wherein said modified nucleobase is a mass-modified nucleobase.

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

12. The primer pair of claim 8, wherein said modified nucleobase is a universal nucleobase.

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

39. A system, comprising:

(a) a mass spectrometer configured to detect one or more molecular masses of an amplification product of claim 13;

(b) a database of known molecular masses and/or known base compositions of amplification products of members of the bacterial class Alphaproteobacter; 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 class Alphaproteobacter.

40. The system of claim 39 wherein said database of known molecular masses and/or known base compositions of amplification products 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;

10;

8, 11;

7, 2;

3, 14;

13, 15;

1, 16;

6, 9;

5, 12;

4, 17;

18, and 19;

20.

14. An isolated amplification product for identification of a member of the bacterial class Alphaproteobacter, said amplification product produced by a process comprising:

a) amplifying nucleic acid of said member of the bacterial class Alphaproteobacter 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 members of the bacterial class Alphaproteobacter 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 members of the bacterial class Alphaproteobacter, said base composition of said intervening region providing a means for identifying said member of the bacterial genus Alphaproteobacter; 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;

10;

8, 11;

7, 2;

3, 14;

13, 15;

1, 16;

6, 9;

5, 12;

4, 17;

18, and 19;

20.

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.

34. A kit comprising one or more purified primer pairs for identifying a member of bacterial class Alphaproteobacter 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;

10;

8, 11;

7, 2;

3, 14;

13, 15;

1, 16;

6, 9;

5, 12;

4,

35. The kit of claim 34 further comprising one or more primers pair targeted to RNAse P.

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;

17;

18, and 19;

20.

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.