Detection of polyketide synthetase gene expression in Karenia brevis

• US 20070111243A1
• Filed: 10/30/2006
• Published: 05/17/2007
• Est. Priority Date: 10/28/2005
• Status: Active Grant

• Current Assignee: University of South Florida (State University System of Florida)
• Original Assignee: University of South Florida (State University System of Florida)
• Inventors: Paul, John
• Granted Patent: US 7,422,857 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The present invention concerns an assay for the detection of polyketide synthetase (PKS) mRNA from the red tide dinoflagellate Karenia brevis. Another aspect of the invention concerns NASBA primers and a molecular beacon that enable detection of mRNA of one of the PKS genes. In another aspect, the present invention includes an internal control RNA (IC-RNA) that enables quantitative detection of the target as well as correction for inhibition of amplification. The assay of the invention provides a rapid and specific detection of the putative toxin-producing genes of the Florida Red Tide Organism. The assay of the invention is useful for determining the virulence of red tide blooms.

Claims

1. A primer specific for polyketide synthetase gene expression from Karenia brevis.

2. The primer of claim 1, wherein the primer is specific for the nucleic acid sequence of SEQ ID NO:

1.

4. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

2.

5. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

3.

6. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

4.

7. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

5.

8. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

6.

9. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

7.

10. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

8.

11. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

10.

12. The isolated polynucleotide of claim 1, which comprises SEQ ID NO:

11.

13. The isolated polynucleotide of claim 1, wherein said nucleic acid sequence consists of SEQ ID NO:

2, 3, 4, 5, 6, 7, 8, 10, or 11, or a functional fragment of any of the foregoing.

3. An isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

2, 3, 4, 5, 6, 7, 8, 10, and 11, or a functional fragment of any of the foregoing.

14. A method for detecting the presence of Karenia brevis in a sample, comprising contacting a polynucleotide region of the polyketide synthetase gene of Karenia brevis with amplification primers;

amplifying the polynucleotide region of the polyketide synthetase gene; and

detecting the presence of an amplified polynucleotide, wherein the presence of the amplified polynucleotide is indicative of the presence of Karenia brevis in the sample.

15. The method of claim 14, wherein the polynucleotide region of the polyketide synthetase gene comprises SEQ ID NO:

1.

16. The method of claim 14, wherein the amplification primers comprise SEQ ID NO:

5 and SEQ ID NO;

6, or a fragment of either or both of the foregoing.

17. The method of claim 14, wherein said method further comprises contacting the polynucleotide region of the polyketide synthetase gene with a labeled molecular beacon.

18. The method of claim 17, wherein the labeled molecular beacon comprises SEQ ID NO:

7 or SEQ ID NO;

8.

19. The method of claim 14, wherein said amplifying comprises nucleic acid sequence based amplification (NASBA).

20. The method of claim 14, wherein said amplifying comprises RT-PCR.

21. The method of claim 14, wherein said detecting comprises hybridization of the amplified product with a probe.

22. The method of claim 14, wherein the sample is salt water.

23. The method of claim 14, wherein said method further comprises determining the quantity of amplified polynucleotide within the sample, the quantity of Karenia brevis cells within the sample, or both.

24. A kit for detecting the presence of Karenia brevis in a sample, comprising two or more isolated polynucleotides selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

10, and SEQ ID NO;

11, or a functional fragment of any of the foregoing.