Detection of ruminant DNA via PCR

• US 20050260618A1
• Filed: 01/28/2005
• Published: 11/24/2005
• Est. Priority Date: 01/30/2004
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Osburn, Bennie, Wong, Alice, Sawyer, Mary, Smith, Wayne, Cullor, James, Rensen, Gabriel
• Application Number:
  US11/046,190
• Publication Number:
  US 20050260618A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms

Abstract

The present invention provides methods, compositions and kits for amplifying, measuring, and or detecting ruminant DNA in samples.

Claims

1. A method of amplifying ruminant DNA in a sample, said method comprising:

contacting nucleic acid from said sample with an RNase, thereby generating RNase-treated nucleic acid; and

amplifying said RNAse-treated nucleic acid using a first ruminant-specific primer and a second-ruminant-specific primer, thereby amplifying ruminant DNA present in said sample and producing an amplified ruminant DNA.

2. The method of claim 1, wherein said nucleic acid is isolated from said animal feed prior to contacting said nucleic acid with an RNase.

3. The method of claim 1, wherein said ruminant DNA is a member selected from the group consisting of:

cattle DNA, sheep DNA, goat DNA, and combinations thereof.

4. The method of claim 1, wherein said RNase is a member selected from the group consisting of:

RNase A, RNase B, RNase D, RNase E, RNase H, RNase I, RNase P, RNase S, RNase T, RNase V, and combinations thereof.

5. The method of claim 1, wherein said RNase-treated nucleic acid is generated by contacting said isolated nucleic acid with said RNase at about 30°

C. to about 40°

C. for about 15 minutes to about 120 minutes.

6. The method of claim 1, wherein said RNase-treated nucleic acid is generated by contacting said isolated nucleic acid with said RNase at about 37°

C. for about 60 minutes.

7. The method of claim 1, wherein said ruminant DNA comprises a mitochondrial DNA sequence.

8. The method of claim 7, wherein said mitochondrial DNA sequence encodes a member selected from the group consisting of:

cytochrome c, cytochrome b, 12S RNA, ATPase subunit 8, ATPase subunit 6, ATP synthetase, subunit 8, and subsequences and combinations thereof.

9. The method of claim 8, wherein said mitochondrial DNA sequence encodes cytochrome b or a subsequence thereof.

10. The method of claim 1, wherein said first ruminant-specific primer and said second ruminant-specific primer are selected from the group consisting of:

SEQ ID NOS;

1 and 2, SEQ ID NOS;

3 and 4, and SEQ ID NOS;

11 and 12.

11. The method of claim 1, further comprising detecting said amplified ruminant DNA.

12. The method of claim 11, wherein detecting said amplified ruminant DNA comprises detecting a fluorescent signal.

13. The method of claim 11, wherein detecting said amplified ruminant DNA comprises contacting said amplified ruminant DNA with an oligonucleotide probe.

14. The method of claim 13, wherein said ruminant DNA is amplified using a first ruminant-specific primer and a second-ruminant-specific primer comprising the sequences set forth in SEQ ID NOS:

11 and 12 and detecting said amplified ruminant DNA comprises contacting the amplified ruminant DNA with oligonucleotide probes comprising the sequences set forth in SEQ ID NOS;

13 and 14.

15. The method of claim 1, further comprising amplifying said amplified ruminant DNA with a third ruminant-specific primer and a fourth-ruminant-specific primer, thereby producing a second amplified ruminant DNA.

16. The method of claim 15, further comprising detecting said second amplified ruminant DNA.

17. The method of claim 1, wherein said sample is a member selected from the group consisting of:

an animal feed, an animal feed component, a cosmetic, a nutraceutical, a vaccine, a colloidal infusion fluid, or combinations thereof.

18. The method of claim 1, wherein said sample is an animal feed.

19. The method of claim 18, wherein said animal feed is cattle feed.

20. The method of claim 19, wherein said cattle feed comprises about 0.5% to about 30% bovine tallow.

21. The method of claim 19, wherein said cattle feed comprises about 1% bovine tallow.

22. The method of claim 1, wherein said sample is an animal feed component.

23. The method of claim 22, wherein said animal feed component is beef tallow.

24. A kit for amplifying ruminant DNA, said kit comprising:

a first pair of ruminant-specific primers;

an RNAse; and

instructions for use.

25. The kit of claim 24, wherein said RNase is a member selected from the group consisting of:

RNase A, RNase B, RNase D, RNase E, RNase H, RNase I, RNase P, RNase S, RNase T, RNase V, and combinations thereof.

26. The kit of claim 24, wherein said first pair of ruminant-specific primers is selected from the group consisting of the sequences set forth in SEQ ID NOS:

1 and 2;

SEQ ID NOS;

3 and 4; and

SEQ ID NOS;

11 and 12.

27. The kit of claim 24, further comprising a second pair of ruminant-specific primers.

28. The kit of claim 27, wherein said first pair of ruminant-specific primers is selected from the group consisting of the sequences set forth in SEQ ID NOS:

1 and 2 and SEQ ID NOS;

3 and 4, and said second pair of ruminant-specific primers is selected from the group consisting of the sequences set forth in SEQ ID NOS;

1 and 2; and

SEQ ID NOS;

3 and 4.

29. The kit of claim 24, further comprising an oligonucleotide probe for detecting an amplified target sequence.

30. The kit of claim 29, wherein the oligonucleotide probe comprises a sequence selected from the group consisting of:

SEQ ID NO;

13 and 14.

31. An isolated nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NOS:

1, 2, 3, 4, 11, 12, 13, or 14.