PARASITE DETECTION VIA ENDOSYMBIONT DETECTION

• US 20110223599A1
• Filed: 03/11/2011
• Published: 09/15/2011
• Est. Priority Date: 03/14/2010
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Crowder, Christopher, Eshoo, Mark W.
• Granted Patent: US 9,758,840 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6893 for protozoa
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides systems, methods, and compositions for identifying a subject as infected with a parasite by detecting nucleic acid from an endosymbiont of the parasite in a sample from the subject. In certain embodiments, the parasite is a nematode that infects humans or dogs (e.g., D. immitis, O. volvulus, W. bancrofti, B. timori, or B. malayi) and the endosymbiont is Wolbachia.

Claims

1. A method of identifying a parasite in source, comprising:

a) providing;

i) a sample from a source suspected of being infected with a parasite, wherein said parasite is associated with an endosymbiont; and

ii) a nucleic acid detection assay configured to detect nucleic acid from said endosymbiont;

b) contacting said sample with said nucleic acid detection assay under conditions such that the presence or absence of said endosymbiont in said sample is determined, wherein said presence of said endosymbiont identifies said source as being infected with said parasite.

2. The method of claim 1, wherein said source is a subject, and further comprising c) diagnosing said subject as being infected with said parasite based on said presence of said endosymbiont in said sample.

3. The method of claim 2, wherein said diagnosing is accomplished without directly detecting the presence of said parasite in said subject.

4. The method of claim 1, wherein said nucleic acid detection assay comprises at least one primer pair, and said contacting generates endosymbiont amplicons using said primer pair under amplification conditions.

5. The method of claim 4, further comprising a step of determining at least a partial base count of at least a subsequence of said endosymbiont amplicons to product base count data.

6. The method of claim 5, further comprising querying a database comprising at least one base count entry corresponding to an identified nucleic acid to produce a match of the base count data with the base count entry, thereby identifying said endosymbiont amplicon as from said endosymbiont.

7. The method of claim 5, wherein said determining at least a partial base count employs mass spectrometry.

8. The method of claim 5, wherein said determining at least a partial base count does not employ mass spectrometry.

9. The method of claim 1, wherein said nucleic acid detection assay is selected from the group consisting of:

a fluorogenic 5′

nuclease assay, a sequencing assay, a polymerase chain reaction assay, a hybridization assay, a microarray assay, a bead array assay, a primer extension assay, an enzyme mismatch cleavage assay, a branched hybridization assay, a rolling circle replication assay, a molecular beacon assay, a cycling probe assay, a ligase chain reaction assay, a sandwich hybridization assay, an invasive cleavage assay, and a Line Probe Assay.

10. The method of claim 1, wherein said parasite is a nematode.

11. The method of claim 10, wherein said nematode infects dogs or cats.

12. The method of claim 10, wherein said nematode infects humans.

13. The method of claim 1, wherein said endosymbiont is a Wolbachia species.

14. The method of claim 13, wherein said parasite is selected from the group consisting of:

Dirofilaria immitis, Onchocerca volvulus, Wuchereria bancrofti, Brugia timori, and Brugia malayi.

15. The method of claim 1, wherein said nucleic acid detection assay comprises at least one primer pair, wherein said primer pair is selected from the group consisting of:

SEQ ID NOs;

1 and 2;

SEQ ID NOs;

3 and 4;

SEQ ID NOs;

5 and 6;

SEQ ID NOs;

7 and 8; and

SEQ ID NOs;

9 and 10.

16. The method of claim 15, wherein said nucleic acid detection assay comprises at least one primer pair, wherein said primer pair is configured to hybridize with conserved regions of said endosymbiont nucleic acid that flank a variable region of said endosymbiont nucleic acid.

17. A system comprising:

a) a sample from a subject suspected of being infected with a parasite, wherein said parasite is associated with an endosymbiont; and

b) a nucleic acid detection assay configured to detect nucleic acid from said endosymbiont.

18. The system of claim 17, wherein said endosymbiont is Wolbachia.

19. The system of claim 17, wherein said parasite is a nematode.

20. The method of claim 19, wherein said nematode infects dogs or cats.

21. The method of claim 19, wherein said nematode infects humans.

22. The method of claim 17, wherein said parasite is selected from the group consisting of:

Dirofilaria immitis, Onchocerca volvulus, Wuchereria bancrofti, Brugia timori, and Brugia malayi.

23. A composition comprising at least one purified oligonucleotide primer pair that comprises forward and reverse primers, wherein said primer pair comprises nucleic acid sequences that are substantially complementary to a nucleic acid sequence from an endosymbiont, wherein said primer pair is configured to hybridize with conserved regions of said nucleic acid from said endosymbiont that flank variable regions of said nucleic acid from said endosymbiont.

24. The composition of claim 23, wherein said forward and reverse primers are about 15 to 35 nucleobases in length, and wherein the forward primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS:

1, 3, 5, 7, and 9, and the reverse primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS;

2, 4, 6, 8, and 10.

25. The composition of claim 23, wherein said primer pair is selected from the group of primer pair sequences consisting of:

SEQ ID NOS;

1;

2, 3;

4, 5;

6, 7;

8, and 9;

10.

26. The composition of claim 23, wherein said primer pair is configured to generate amplicons that are 45 to 200 nucleobases in length.

27. The composition of claim 23, wherein said forward and/or reverse primer comprises at least one molecular mass modifying tag.

28. The composition of claim 23, wherein said forward and/or reverse primer comprises at least one modified nucleobase.

30. The composition of claim 28, wherein said modified nucleobase is a mass modified nucleobase.

31. The composition of claim 30, wherein said mass modified nucleobase is 5-Iodo-C.

32. The composition of claim 28, wherein said modified nucleobase is a universal nucleobase.

33. The composition of claim 32, wherein said universal nucleobase is inosine.

34. The composition of claim 23, wherein said endosymbiont parasite is selected from the group consisting of:

Wolbachia endosymbiont of Dirofilaria immitis, Wolbachia endosymbiont of Onchocerca volvulus, Wolbachia endosymbiont of Brugia malayi, Wolbachia endosymbiont of Wuchereria bancrofti, and Wolbachia endosymbiont of Brugia timori.

29. The composition of claim 29, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

35. A composition comprising an isolated primer 15-35 bases in length selected from the group consisting of SEQ ID NOs 1-10.

36. A kit, comprising at least one purified oligonucleotide primer pair that comprises forward and reverse primers that are about 20 to 35 nucleobases in length, and wherein said forward primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS:

1, 3, 5, 7, and 9, and said reverse primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS;

2, 4, 6, 8, and 10.

37. A system, comprising:

(a) a mass spectrometer configured to detect one or more molecular masses of amplicons produced using at least one purified oligonucleotide primer pair that comprises forward and reverse primers, wherein said primer pair comprises nucleic acid sequences that are substantially complementary to nucleic acid sequences from an endosymbiont; and

(b) a controller operably connected to said mass spectrometer, said controller configured to correlate said molecular masses of said amplicons with one or more endosymbiont identities.

38. The system of claim 37, wherein said forward and reverse primers are about 15 to 35 nucleobases in length, and wherein the forward primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS:

1, 3, 5, 7, and 9, and the reverse primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS;

2, 4, 6, 8, and 10.

39. A method of detecting a parasite associated with an endosymbiont, the method comprising detecting a presence of said endosymbiont in a sample, thereby detecting said parasite associated with said endosymbiont.

40. The method of claim 39, wherein said detecting comprises detecting nucleic acid from said endosymbiont using a nucleic acid detection assay.

41. A method of detecting an endosymbiont associated with a parasite, the method comprising detecting a presence of said parasite in a sample, thereby detecting said endosymbiont associated with said parasite.

42. The method of claim 41, wherein said detecting comprises detecting nucleic acid from said parasite using a nucleic acid detection assay.