Parasite detection via endosymbiont detection

• US 9,758,840 B2
• Filed: 03/11/2011
• Issued: 09/12/2017
• Est. Priority Date: 03/14/2010
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Eshoo, Mark W., Crowder, Christopher
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/046,186
• Publication Number:
  US 20110223599A1
• Time in Patent Office: 2,377 Days
• Field of Search: None
• 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 a subject comprising:

a) providing;

i) a blood sample from a human subject suspected of being infected with a parasite, wherein said parasite is associated with a specific Wolbachia species or subspecies endosymbiont; and

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

b) contacting said sample with said nucleic acid detection assay under conditions such that the presence or absence of said Wolbachia species or subspecies endosymbiont in said sample is determined, wherein said presence of said Wolbachia species or subspecies endosymbiont uniquely identifies said subject as being infected with said parasite;

c) determining a partial base count of at least a subsequence of said endosymbiont amplicons to produce base count data; and

d) diagnosing said subject as being infected with said parasite based on said presence of said Wolbachia species or subspecies endosymbiont in said sample wherein said diagnosing is accomplished without directly detecting the presence of said parasite in said subject.

2. The method of claim 1, 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.

3. The method of claim 1, wherein said determining a partial base count employs mass spectrometry.

4. The method of claim 1, wherein said determining a partial base count does not employ mass spectrometry.

5. 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.

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

7. The method of claim 6, wherein said nematode infects dogs or cats.

8. The method of claim 6, wherein said nematode infects humans.

9. The method of claim 1, wherein said parasite is selected from the group consisting of:

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

10. 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.

11. The method of claim 10, 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.

12. 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 is configured to hybridize with conserved regions of Wolbachia nucleic acid that flank a variable region of nucleic acid specific for a Wolbachia species or subspecies endosymbiont;

b) a controller operably connected to said mass spectrometer, said controller configured to correlate said molecular masses of said amplicons with one or more Wolbachiaspecies or subspecies endosymbiont identities; and

c) a database of partial base counts wherein said database of partial base counts comprises partial base counts comprising the number of any one of four nucleobase types, any two of four nucleobase types, or any three of four nucleobase types.