Compositions for use in identification of papillomavirus

• US 8,084,207 B2
• Filed: 10/01/2009
• Issued: 12/27/2011
• Est. Priority Date: 03/03/2005
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Sampath, Rangarajan, Blyn, Lawrence B., Kreft, Rachael, Hall, Thomas A., Li, Feng
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US12/571,925
• Publication Number:
  US 20100136515A1
• Time in Patent Office: 817 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The present invention relates generally to identification of HPV, 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 purified oligonucleotides, wherein the oligonucleotides consist of a primer pair that consists of a forward and a reverse primer, wherein the forward and reverse primers consist of sequences selected from the group of primer pair sequences consisting of SEQ ID NOS:

1;

9, 2;

10, 3;

11, 4;

12, 5;

13, 6;

14, 7;

15, and 8;

16.

2. A kit comprising the composition of claim 1.

3. The composition of claim 1, wherein said forward and/or reverse primer comprises at least one molecular mass modifying tag.

4. The composition of claim 1, wherein said forward and/or reverse primer comprises at least one modified nucleobase.

5. The composition of claim 4, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

6. The composition of claim 4, wherein said modified nucleobase is a mass modified nucleobase.

7. The composition of claim 6, wherein said mass modified nucleobase is 5-Iodo-C.

8. The composition of claim 4, wherein said modified nucleobase is a universal nucleobase.

9. The composition of claim 8, wherein said universal nucleobase is inosine.

10. A method of determining the presence of a HPV in at least one sample, the method comprising:

(a) amplifying one or more segments of at least one nucleic acid from said sample using the primer pair of claim 1 to produce at least one amplification product; and

(b) detecting said amplification product, thereby determining said presence of said HPV in said sample.

11. The method of claim 10, wherein (a) comprises amplifying said one or more segments of said at least one nucleic acid from at least two samples obtained from different geographical locations to produce at least two amplification products, and (b) comprises detecting said amplification products, thereby tracking an epidemic spread of said HPV.

12. The method of claim 10, wherein (b) comprises determining an amount of said HPV in said sample.

13. The method of claim 10, wherein (b) comprises detecting a molecular mass of said amplification product.

14. The method of claim 10, wherein (b) comprises determining a base composition of said amplification product, wherein said base composition identifies the number of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof in said amplification product, whereby said base composition indicates the presence of HPV in said sample or identifies said HPV in said sample.

15. The method of claim 14, comprising comparing said base composition of said amplification product to calculated or measured base compositions of amplification products of one or more known HPV present in a database with the proviso that sequencing of said amplification product is not used to indicate the presence of or to identify said HPV, wherein a match between said determined base composition and said calculated or measured base composition in said database indicates the presence of or identifies said HPV.

16. A method of identifying one or more HPV bioagents in a sample, the method comprising:

(a) amplifying two or more segments of a nucleic acid from said one or more HPV bioagents in said sample with two or more oligonucleotide primer pairs of claim 1 to obtain two or more amplification products;

(b) determining two or more molecular masses and/or base compositions of said two or more amplification products; and

(c) comparing said two or more molecular masses and/or said base compositions of said two or more amplification products with known molecular masses and/or known base compositions of amplification products of known HPV bioagents produced with said two or more primer pairs to identify said one or more HPV bioagents in said sample.

17. The method of claim 16, comprising identifying said one or more HPV bioagents in said sample using three, four, five, six, seven, eight or more primer pairs.

18. The method of claim 16, wherein said one or more HPV bioagents in said sample cannot be identified using a single primer pair of said two or more primer pairs.

19. The method of claim 16, comprising obtaining said two or more molecular masses of said two or more amplification products via mass spectrometry.

20. The method of claim 16, comprising calculating said two or more base compositions from said two or more molecular masses of said two or more amplification products.

21. The method of claim 16, wherein said HPV bioagents are selected from the group consisting of a Papillomaviridae family, a genus thereof, a species thereof, a sub-species thereof, and combinations thereof.

22. The method of claim 16, wherein said determining said two or more molecular masses and/or base compositions is conducted without sequencing said two or more amplification products.

23. The method of claim 16, wherein said one or more HPV bioagents in said sample cannot be identified using a single primer pair of said two or more primer pairs.

24. The method of claim 16, wherein said one or more HPV bioagents in a sample are identified by comparing three or more molecular masses and/or base compositions of three or more amplification products with a database of known molecular masses and/or known base compositions of amplification products of known HPV bioagents produced with said three or more primer pairs.

25. The method of claim 16, wherein said two or more segments of said nucleic acid are amplified from a single gene.

26. The method of claim 16, wherein said two or more segments of said nucleic acid are amplified from different genes.

27. The method of claim 16, wherein members of said primer pairs hybridize to conserved regions of said nucleic acid that flank a variable region.

28. The method of claim 27, wherein said variable region varies between at least two of said HPV bioagents.

29. The method of claim 27, wherein said variable region uniquely varies between at least five of said HPV bioagents.

30. The method of claim 16, wherein said two or more amplification products obtained in (a) comprise major classification and subgroup identifying amplification products.

31. The method of claim 30, comprising comparing said molecular masses and/or said base compositions of said two or more amplification products to calculated or measured molecular masses or base compositions of amplification products of known HPV bioagents in a database comprising genus specific amplification products, species specific amplification products, strain specific amplification products or nucleotide polymorphism specific amplification products produced with said two or more oligonucleotide primer pairs, wherein one or more matches between said two or more amplification products and one or more entries in said database identifies said one or more HPV bioagents, classifies a major classification of said one or more HPV bioagents, and/or differentiates between subgroups of known and unknown HPV bioagents in said sample.

32. The method of claim 31, wherein said major classification of said one or more HPV bioagents comprises a genus or species classification of said one or more HPV bioagents.

33. The method of claim 31, wherein said subgroups of known and unknown HPV bioagents comprise family, strain and nucleotide variations of said one or more HPV bioagents.