Detection of contaminating DNA via apmplification of sequences of less than 100 bp

• US 20070269822A1
• Filed: 02/02/2007
• Published: 11/22/2007
• Est. Priority Date: 02/03/2006
• 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: Smith, Wayne, Cullor, James, Osburn, Bennie, Rensen, Gabriel
• Application Number:
  US11/701,596
• Publication Number:
  US 20070269822A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Abstract

The present invention provides methods, compositions and kits for amplifying, measuring, and/or detecting contaminating DNA (e.g., ruminant and non-ruminant) in samples by amplification and/or detection of a target DNA of less than 100 base pairs in the samples.

Claims

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

amplifying nucleic acid from the sample using a pair of primers designed to amplify a target DNA sequence of less than about 100 base pairs, thereby amplifying the contaminating DNA present in said sample.

2. The method of claim 1, wherein the target sequence is about 60 to about 100 base pairs.

3. The method of claim 1, wherein the target sequence is about 70 to about 90 base pairs.

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

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

5. The method of claim 1, wherein said contaminating DNA comprises a mitochondrial DNA sequence.

6. The method of claim 5, 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.

7. The method of claim 5, wherein said mitochondrial DNA sequence encodes cytochrome b or a subsequence thereof.

8. The method of claim 1, wherein said pair of primers comprises sequences selected from the group consisting of:

SEQ ID NOS;

1 and 2;

SEQ ID NOS;

5 and 6;

SEQ ID NOS;

11 and 12;

SEQ ID NOS;

15 and 16; and

SEQ ID NOS;

19 and 20.

9. The method of claim 1, further comprising detecting said amplified contaminating DNA.

10. The method of claim 9, wherein detecting said amplified contaminating DNA comprises contacting said amplified contaminating DNA with an oligonucleotide probe.

11. The method of claim 10, wherein said oligonucleotide probe is labeled with a detectable label.

12. The method of claim 10, further comprising contacting said amplified contaminating DNA with a second oligonucleotide probe.

13. The method of claim 12, wherein detecting said amplified contaminating DNA comprises contacting said amplified contaminating DNA with a first oligonucleotide probe linked to a donor fluorophore and a second oligonucleotide probe linked to an acceptor fluorophore, wherein the donor fluorophore and the acceptor fluorophore interact to generate a detectable signal.

14. The method of claim 13, wherein said first oligonucleotide probe and said second oligonucleotide probe comprise the sequences set forth in SEQ ID NOS:

3 and 4;

SEQ ID NOS;

7 and 8;

SEQ ID NOS;

13 and 14, SEQ I) NOS;

17 and 18;

or SEQ ID NOS;

21 and 22.

15. 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.

16. The method of claim 1, wherein said animal feed is cattle feed.

17. The method of claim 16, wherein said cattle feed comprises bovine tallow.

18. A kit for amplifying contaminating DNA, said kit comprising:

a pair of primers designed to amplify a target DNA sequence of less than about 100 base pairs, wherein the pair of primers is selected from the group consisting of SEQ ID NOS;

1 and 2;

SEQ ID NOS;

5 and 6;

SEQ ID NOS;

11 and 12;

SEQ ID NOS;

15 and 16; and

SEQ ID NOS;

19 and 20; and

instructions for use.

19. The kit of claim 18, further comprising a first oligonucleotide probe linked to a donor fluorophore and a second oligonucleotide probe linked to an acceptor fluorophore, wherein the donor fluorophore and the acceptor fluorophore interact to generate a detectable signal.

20. The kit of claim 19, wherein said first oligonucleotide probe and said second oligonucleotide probe comprise:

SEQ ID NOS;

3 and 4;

SEQ ID NOS;

7 and 8;

SEQ ID NOS;

13 and 14, SEQ ID NOS;

17 and 18;

or SEQ ID NOS;

21 and 22.

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

1, 2, 3, 4, 5, 6, 7, 8;

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.