Method to assess quorum sensing potential of microbial communities

• US 20040023254A1
• Filed: 01/07/2003
• Published: 02/05/2004
• Est. Priority Date: 01/08/2002
• Status: Abandoned Application

• Current Assignee: James A. Romesser, Jeffry J. Fuhrmann
• Original Assignee: James A. Romesser, Jeffry J. Fuhrmann
• Inventors: Fuhrmann, Jeffry J., Romesser, James A.
• Application Number:
  US10/338,110
• Publication Number:
  US 20040023254A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important

Abstract

A method for detecting quorum sensing potential in a sample is provided comprising extracting nucleic acid from a sample comprising at least one type of microorganism; performing a polymerase chain reaction on the nucleic acid using oligonucleotide primers, wherein each of the primers comprises at least 15 nucleobases and anneal to a consensus sequence of a lux gene and homologs thereof, wherein the first eight nucleobases at the 3′ end of the primers comprise a match to said consensus sequence with optional degeneracy at positions 4-8 from the 3′ end of the primers; separating the amplified DNA fragments; and determining whether any amplified DNA fragment corresponds to the size of a predicted product related to quorum sensing potential. Methods of amplifying fragments of lux genes and homologs thereof are also provided as well as isolated nucleic acid fragments of lux and kits for performing PCR reactions.

Claims

1. A method of detecting quorum sensing potential of a microorganism in a sample comprising:

(a) extracting nucleic acid from a sample comprising at least one type of microorganism;

(b) performing a polymerase chain reaction using said nucleic acid wherein said polymerase chain reaction comprises a first oligonucleotide primer and a second oligonucleotide primer, wherein each of said primers comprises at least 15 nucleobases, wherein said primers anneal to a consensus sequence of a lux gene and homologs thereof, wherein the first eight nucleobases at the 3′

end of said primers comprise a match to said consensus sequence with optional degeneracy at positions 4-8 from the 3′

end of said primers;

wherein said polymerase chain reaction results in amplified DNA fragments;

(c) separating said amplified DNA fragments; and

(d) determining whether any amplified DNA fragment corresponds to the size of a predicted product related to quorum sensing potential, thereby detecting quorum sensing potential of said microorganism in said sample.

2. The method of claim 1 wherein said lux gene is selected from the group consisting of luxI, luxR, and luxS.

3. The method of claim 1 wherein said microorganism is a bacterium.

4. The method of claim 3 wherein said bacterium is a Gram-negative bacterium.

5. The method of claim 1 wherein said sample is taken from the environment.

6. The method of claim 1 wherein said separating is performed on a gel.

7. The method of claim 1 wherein said separating is performed by liquid chromatography.

8. The method of claim 2 wherein said lux gene is the luxI gene.

9. The method of claim 8 wherein said first oligonucleotide primer comprises SEQ ID NO:

1 and said second oligonucleotide primer comprises SEQ ID NO;

2.

10. The method of claim 8 wherein said first oligonucleotide primer comprises SEQ ID NO:

3 and said second oligonucleotide primer comprises SEQ ID NO;

4.

11. The method of claim 8 wherein said first oligonucleotide primer comprises SEQ ID NO:

5 and said second oligonucleotide primer comprises SEQ ID NO;

6.

12. The method of claim 8 wherein said first oligonucleotide primer comprises SEQ ID NO:

7 and said second oligonucleotide primer comprises SEQ ID NO;

8.

13. The method of claim 1 wherein said lux gene is the luxS gene.

14. The method of claim 13 wherein said first oligonucleotide primer comprises SEQ ID NO:

9 and said second oligonucleotide primer comprises SEQ ID NO;

10.

15. A composition comprising

(a) a nucleic acid sequence comprising a lux gene or homolog thereof;

(b) at least two oligonucleotide primers, wherein each of said primers comprises at least 15 nucleobases, wherein said primers anneal to a consensus sequence of a lux gene and homologs thereof;

wherein the first eight nucleobases at the 3′

end of said primers comprise a match to said consensus sequence with optional degeneracy at positions 4-8 from the 3′

end of said primers;

(c) at least one enzyme having DNA polymerase activity.

16. The composition of claim 15 further comprising a mixture of deoxynucleotide triphosphates.

17. The composition of claim 15 wherein said primers comprise the sequences of SEQ ID NO:

1 and SEQ ID NO;

2.

18. The composition of claim 15 wherein said primers comprise the sequences of SEQ ID NO:

3 and SEQ ID NO;

4.

19. The composition of claim 15 wherein said primers comprise the sequences of SEQ ID NO:

5 and SEQ ID NO;

6.

20. The composition of claim 15 wherein said primers comprise the sequences of SEQ ID NO:

7 and SEQ ID NO;

8.

21. The composition of claim 15 wherein said primers comprise at least one pair of primers selected from the group consisting of SEQ ID NO:

1 and SEQ ID NO;

2;

SEQ ID NO;

3 and SEQ ID NO;

4;

SEQ ID NO;

5 and SEQ ID NO;

6;

SEQ ID NO;

7 and SEQ ID NO;

8; and

SEQ ID NO;

9 and SEQ ID NO;

10.

22. A method of amplifying a fragment of a lux gene or homolog thereof comprising:

(a) extracting nucleic acid from a sample comprising at least one type of microorganism, said nucleic acid comprising a lux gene or homolog thereof;

(b) performing a polymerase chain reaction using said nucleic acid wherein said polymerase chain reaction comprises a first oligonucleotide primer and a second oligonucleotide primer, wherein each of said primers comprises at least 15 nucleobases, wherein said primers anneal to a consensus sequence of a lux gene and homologs thereof;

wherein the first eight nucleobases at the 3′

end of said primers comprise a match to said consensus sequence with optional degeneracy at positions 4-8 from the 3′

end of said primers;

wherein said polymerase chain reaction results in amplified DNA fragments.

23. The method of claim 22 further comprising isolating an amplified fragment of said lux gene or homolog thereof.

24. The method of claim 22 wherein said primers are selected from the group consisting of SEQ ID NO:

1 and SEQ ID NO;

2;

SEQ ID NO;

3 and SEQ ID NO;

4;

SEQ ID NO;

5 and SEQ ID NO;

6;

SEQ ID NO;

7 and SEQ ID NO;

8; and

SEQ ID NO;

9 and SEQ ID NO;

10.

25. The isolated amplified fragment of claim 23.

26. The method of claim 24 further comprising isolating an amplified fragment of said lux gene or homolog thereof.

27. The isolated amplified fragment of claim 26.

28. A kit for the amplification of a portion of a lux gene, or homolog thereof comprising, in separate containers:

(a) a polymerase,

(b) a plurality of deoxynucleotide triphosphates;

(c) a first oligonucleotide primer; and

(d) a second oligonucleotide primer;

wherein each of said primers comprises at least 15 nucleobases, wherein said primers anneal to a consensus sequence of a lux gene and homologs thereof; and

wherein the first eight nucleobases at the 3′

end of said primers comprise a match to said consensus sequence with optional degeneracy at positions 4-8 from the 3′

end of said primers.

29. The kit of claim 28 wherein said kit first oligonucleotide primer comprises SEQ ID NO:

1 and said second oligonucleotide primer comprises SEQ ID NO;

2.

30. The kit of claim 28 wherein said kit first oligonucleotide primer comprises SEQ ID NO:

3 and said second oligonucleotide primer comprises SEQ ID NO;

4.

31. The kit of claim 28 wherein said kit first oligonucleotide primer comprises SEQ ID NO:

5 and said second oligonucleotide primer comprises SEQ ID NO;

6.

32. The kit of claim 28 wherein said kit first oligonucleotide primer comprises SEQ ID NO:

7 and said second oligonucleotide primer comprises SEQ ID NO;

8.

33. The kit of claim 28 wherein said kit first oligonucleotide primer comprises SEQ ID NO:

9 and said second oligonucleotide primer comprises SEQ ID NO;

10.