Detection and quantification of aromatic oxygenase genes by real-time PCR

• US 20040161767A1
• Filed: 06/30/2003
• Published: 08/19/2004
• Est. Priority Date: 06/28/2002
• Status: Abandoned Application

• Current Assignee: Purdue Research Foundation (Purdue University)
• Original Assignee: Purdue Research Foundation (Purdue University)
• Inventors: Baldwin, Brett R., Nakatsu, Cindy H., Nies, Loring F.
• Application Number:
  US10/611,089
• Publication Number:
  US 20040161767A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention provides a direct manner of assessing the bioremediation potential of microbes in a soil sample by detecting and enumerating the microbes that have the necessary functional genes to metabolize specific pollutants. In particular, the present invention provides novel compositions and methods for analyzing a sample containing a diverse population of microbes to detect and quantify the presence of specific functional aromatic pollutant oxygenase genotypes in the population. The detection and quantification of genotypes in accordance with the invention provides a manner in which the bioremediation potential of the sample can be assessed in a reliable manner. Quantification is achieved in accordance with the invention by quantitative PCR amplification using primers that are constructed or selected to amplify target regions identified to be included in conserved regions of genes from diverse microbial species that have aromatic pollutant metabolism functionality.

Claims

1. A method for assessing the bioremediation potential of a microbial community in a soil or water sample, comprising:

providing a plurality of PCR primer sets, wherein each set corresponds to a distinct family or subfamily of functional aromatic oxygenase genes and is effective to selectively amplify target regions from diverse aromatic oxygenase genes in the corresponding family or subfamily;

providing a mixture of polynucleotides isolated from microbes present in a soil or water sample;

performing one or more quantitative PCR amplification reactions using the primer sets to quantify diverse aromatic oxygenase genes of each corresponding family or subfamily in the mixture; and

determining the bioremediation potential of microbes in the sample based upon results of the one or more quantitative PCR reactions.

2. The method in accordance with claim 1 wherein the sample is contaminated with a plurality of aromatic pollutants.

3. The method in accordance with claim 1 wherein the sample is a sample from a petroleum contaminated site.

4. The method in accordance with claim 1 wherein the plurality of primer sets includes at least two primer sets corresponding to two families or subfamilies of functional aromatic oxygenase genes that encode enzymes having specificity for different aromatic pollutant compounds.

5. The method in accordance with claim 1 wherein said performing comprises performing real-time quantitative PCR analysis.

6. The method in accordance with claim 5 wherein the real-time quantitative PCR analysis is performed using a double stranded DNA-binding dye.

7. The method in accordance with claim 6 wherein the dye is a SYBR Green dye.

8. The method in accordance with claim 5 wherein the real-time quantitative PCR analysis is performed using a member selected from the group consisting of molecular beacons, hybridization probes and hydrolysis probes;

and wherein the member is effective to hybridize to a polynucleotide segment of from about 10 to about 40 bases that is conserved in the members of each family or subfamily.

9. The method in accordance with claim 1 wherein the plurality of primer sets includes at least two primer sets corresponding to two families or subfamilies of functional aromatic oxygenase genes that encode enzymes having specificity for the same aromatic pollutant compound.

10. The method in accordance with claim 1 wherein at least one of the one or more quantitative PCR amplification reactions comprises a multiplex real-time quantitative PCR reaction.

11. The method in accordance with claim 10 wherein the plurality of primer sets includes a first primer set that is effective to selectively amplify a family or subfamily of phenol monooxygenase genes and a second primer set that is effective to selectively amplify a family or subfamily of naphthalene dioxygenase genes;

and wherein the first and second primer sets are used together to amplify diverse target regions in a multiplex real-time quantitative PCR reaction.

12. The method in accordance with claim 11 wherein the first primer set comprises a forward primer having the nucleotide sequence of SEQ ID NO:

18 and a reverse primer having the nucleotide sequence of SEQ ID NO;

19.

13. The method in accordance with claim 11 wherein the second primer set comprises a forward primer having the nucleotide sequence of SEQ ID NO:

1 and a reverse primer having the nucleotide sequence of SEQ ID NO;

2.

14. The method in accordance with claim 10 wherein the plurality of primer sets includes a first primer set that is effective to selectively amplify a family or subfamily of xylene monooxygenase genes and a second primer set that is effective to selectively amplify a family or subfamily of toluene dioxygenase genes;

and wherein the first and second primer sets are used together to amplify diverse target regions in a multiplex real-time quantitative PCR reaction.

15. The method in accordance with claim 14 wherein the first primer set comprises a forward primer having the nucleotide sequence of SEQ ID NO:

5 and a reverse primer having the nucleotide sequence of SEQ ID NO;

6.

16. The method in accordance with claim 14 wherein the second primer set comprises a forward primer having the nucleotide sequence of SEQ ID NO:

3 and a reverse primer having the nucleotide sequence of SEQ ID NO;

4.

17. The method in accordance with claim 10 wherein the plurality of primer sets includes a first primer set that is effective to selectively amplify a first subfamily of biphenyl dioxygenase genes and a second primer set that is effective to selectively amplify a second subfamily of biphenyl dioxygenase genes;

and wherein the first and second primer sets are used together to amplify diverse target regions in a multiplex real-time quantitative PCR reaction.

18. The method in accordance with claim 17 wherein the first primer set comprises a forward primer having the nucleotide sequence of SEQ ID NO:

9 and a reverse primer having the nucleotide sequence of SEQ ID NO;

10.

19. The method in accordance with claim 17 wherein the second primer set comprises a forward primer having the nucleotide sequence of SEQ ID NO:

12 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13.

20. The method in accordance with claim 1 wherein the plurality of primer sets includes at least two primer sets, each of which is effective to selectively amplify a family or subfamily of functional aromatic oxygenase genes selected from the group consisting of naphthalene dioxygenase genes, toluene dioxygenase genes, xylene monooxygenase genes, biphenyl dioxygenase genes, toluene monooxygenase genes and phenol monooxygenase genes.

21. The method in accordance with claim 1 wherein the plurality of primer sets includes at least one primer set selected from the group consisting of:

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

1 and a reverse primer having the nucleotide sequence of SEQ ID NO;

2;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

3 and a reverse primer having the nucleotide sequence of SEQ ID NO;

4;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

5 and a reverse primer having the nucleotide sequence of SEQ ID NO;

6;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

7 and a reverse primer having the nucleotide sequence of SEQ ID NO;

8;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

9 and a reverse primer having the nucleotide sequence of SEQ ID NO;

10;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

11 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

12 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

14 and a reverse primer having the nucleotide sequence of SEQ ID NO;

15;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

16 and a reverse primer having the nucleotide sequence of SEQ ID NO;

17; and

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

18 and a reverse primer having the nucleotide sequence of SEQ ID NO;

19.

22. The method in accordance with claim 1 wherein the plurality of primer sets includes at least two primer sets selected from the group consisting of:

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

1 and a reverse primer having the nucleotide sequence of SEQ ID NO;

2;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

3 and a reverse primer having the nucleotide sequence of SEQ ID NO;

4;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

5 and a reverse primer having the nucleotide sequence of SEQ ID NO;

6;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

7 and a reverse primer having the nucleotide sequence of SEQ ID NO;

8;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

9 and a reverse primer having the nucleotide sequence of SEQ ID NO;

10;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

11 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

12 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

14 and a reverse primer having the nucleotide sequence of SEQ ID NO;

15;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

16 and a reverse primer having the nucleotide sequence of SEQ ID NO;

17; and

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

18 and a reverse primer having the nucleotide sequence of SEQ ID NO;

19.

23. The method in accordance with claim 1 wherein said performing comprises performing real-time quantitative PCR analysis of the mixture using each of the following primer sets:

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

1 and a reverse primer having the nucleotide sequence of SEQ ID NO;

2;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

3 and a reverse primer having the nucleotide sequence of SEQ ID NO;

4;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

5 and a reverse primer having the nucleotide sequence of SEQ ID NO;

6;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

7 and a reverse primer having the nucleotide sequence of SEQ ID NO;

8;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

9 and a reverse primer having the nucleotide sequence of SEQ ID NO;

10;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

11 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

12 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

14 and a reverse primer having the nucleotide sequence of SEQ ID NO;

15;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

16 and a reverse primer having the nucleotide sequence of SEQ ID NO;

17; and

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

18 and a reverse primer having the nucleotide sequence of SEQ ID NO;

19.

24. A screening protocol for detecting and quantifying multiple families or subfamilies of functional aromatic oxygenase genes of diverse aromatic pollutant-degrading microbial species in a soil or water sample, comprising:

providing a mixture of polynucleotides isolated from microbes present in a soil or water sample; and

performing quantitative PCR analysis of the mixture using a plurality of primer sets configured to selectively amplify different families or subfamilies of functional aromatic oxygenase genes.

25. The protocol in accordance with claim 24 wherein a plurality of the primer sets are suitable for use together in a multiplex real-time PCR reaction.

26. The protocol in accordance with claim 24 wherein each of the primer sets is used in separate real-time quantitative PCR reactions to separately quantify each corresponding family or subfamily of functional aromatic oxygenase genes.

27. A method of monitoring the bioremediation potential of a microbial community in a soil or water system contaminated with aromatic pollutants, comprising:

providing a mixture of polynucleotides isolated from a soil or water sample corresponding to the system; and

performing quantitative PCR analysis of said mixture using a plurality of primer sets configured to selectively amplify target segments from corresponding families or subfamilies of aromatic oxygenase genes to provide a quantity value corresponding to aromatic oxygenase gene abundance in the sample;

wherein the aromatic oxygenase gene abundance correlates with the bioremediation potential of the sample.

28. The method in accordance with claim 27, further comprising perturbing the system, waiting a period of time sufficient to allow the microbial community in the system to respond to said perturbing, and repeating said providing and performing to determine whether the bioremediation potential of the sample has changed.

29. The method in accordance with claim 27 wherein said quantitative PCR is competitive, noncompetitive, kinetic, or combinations thereof.

30. The method in accordance with claim 27 wherein the mixture of polynucleotides comprises a mixture of RNA polynucleotides and wherein said performing comprises performing quantitative RT-PCR on said RNA using a plurality of primer sets configured to selectively amplify target segments from corresponding families or subfamilies of mRNA corresponding to aromatic oxygenase genes to provide a quantity value corresponding to aromatic oxygenase gene expression in the sample.

31. The method in accordance with claim 30 wherein said quantitative RT-PCR is competitive, noncompetitive, kinetic, or combinations thereof.

32. A real-time Polymerase Chain Reaction (PCR) method for the selective detection and quantification of diverse families or subfamilies of aromatic oxygenase genes, each family or subfamily including a unique conserved region or a plurality of unique conserved sub-regions, said method comprising:

providing a mixture of polynucleotides isolated from a soil or water sample;

providing a plurality of primer sets configured to selectively amplify target segments from corresponding families or subfamilies of aromatic oxygenase genes and

performing quantitative PCR analysis of said mixture using the plurality of primer sets to provide a quantity value corresponding to aromatic oxygenase gene abundance in the sample.

33. The real-time PCR method in accordance with claim 32 wherein the PCR method comprises at least one polymerization reaction performed by adding two or more primer sets to the same PCR mixture, each primer set being specific for a single family or subfamily of aromatic oxygenase genes.

34. The real-time PCR method according to claim 32 wherein each of the primer sets is effective for amplifying a target segment from a different family or subfamily including aromatic oxygenase genes selected from the group consisting of a naphthalene dioxygenase genes, toluene dioxygenase genes, xylene monooxygenase genes, biphenyl dioxygenase genes, toluene monooxygenase genes and phenol monooxygenase genes.

35. The real-time PCR method according to claim 32 wherein at least one of the primer sets is selected from the group consisting of:

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

1 and a reverse primer having the nucleotide sequence of SEQ ID NO;

2;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

3 and a reverse primer having the nucleotide sequence of SEQ ID NO;

4;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

5 and a reverse primer having the nucleotide sequence of SEQ ID NO;

6;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

7 and a reverse primer having the nucleotide sequence of SEQ ID NO;

8;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

9 and a reverse primer having the nucleotide sequence of SEQ ID NO;

10;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

11 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

12 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

14 and a reverse primer having the nucleotide sequence of SEQ ID NO;

15;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

16 and a reverse primer having the nucleotide sequence of SEQ ID NO;

17; and

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

18 and a reverse primer having the nucleotide sequence of SEQ ID NO;

19.

36. The real-time PCR method according claim 32 wherein said PCR is a reverse-transcription (RT) PCR.

37. The real-time PCR method according to claim 32 wherein in the same one-tube reaction a standard nucleic acid sequence is simultaneously amplified and quantified according to real-time PCR principles;

and wherein the standard nucleic acid sequence is added in a known copy number to a sample to be tested.

38. The real-time PCR method according to claim 32 wherein a double stranded DNA-binding dye is added to a sample to be tested.

39. The real-time PCR method according to claim 38 wherein primer sets corresponding to two or more families or subfamilies of aromatic oxygenase genes are included a single one-tube reaction for simultaneous amplification and quantification of the two or more families or subfamilies.

40. A primer set selected from the group consisting of:

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

1 and a reverse primer having the nucleotide sequence of SEQ ID NO;

2;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

3 and a reverse primer having the nucleotide sequence of SEQ ID NO;

4;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

5 and a reverse primer having the nucleotide sequence of SEQ ID NO;

6;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

7 and a reverse primer having the nucleotide sequence of SEQ ID NO;

8;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

9 and a reverse primer having the nucleotide sequence of SEQ ID NO;

10;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

11 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

12 and a reverse primer having the nucleotide sequence of SEQ ID NO;

13;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

14 and a reverse primer having the nucleotide sequence of SEQ ID NO;

15;

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

16 and a reverse primer having the nucleotide sequence of SEQ ID NO;

17; and

a set comprising a forward primer having the nucleotide sequence of SEQ ID NO;

18 and a reverse primer having the nucleotide sequence of SEQ ID NO;

19.

41. A primer set pair for performing multiplex real-time quantitative PCR comprising a forward primer having the nucleotide sequence of SEQ ID NO:

18, a reverse primer having the nucleotide sequence of SEQ ID NO;

19, a forward primer having the nucleotide sequence of SEQ ID NO;

1, and a reverse primer having the nucleotide sequence of SEQ ID NO;

2.

42. A primer set pair for performing multiplex real-time quantitative PCR comprising a forward primer having the nucleotide sequence of SEQ ID NO:

5, a reverse primer having the nucleotide sequence of SEQ ID NO;

6, a forward primer having the nucleotide sequence of SEQ ID NO;

3, and a reverse primer having the nucleotide sequence of SEQ ID NO;

4.

43. A primer set pair for performing multiplex real-time quantitative PCR comprising a forward primer having the nucleotide sequence of SEQ ID NO:

9, a reverse primer having the nucleotide sequence of SEQ ID NO;

10, a forward primer having the nucleotide sequence of SEQ ID NO;

12, a reverse primer having the nucleotide sequence of SEQ ID NO;

13.

44. A method for making a series of PCR primer sets for use in determining bioremediation potential of microbes in a sample to be analyzed, comprising:

identifying a plurality of aromatic pollutants for which bioremediation potential is to be determined;

preparing an alignment of functional aromatic oxygenase genes for each group of oxygenase genes having specificity for one of the pollutants;

wherein each of the alignments includes genes from diverse species that encode oxygenase enzymes effective to oxygenate the corresponding aromatic pollutant;

identifying a region of each alignment comprising from about 50 to about 1000 bases that is substantially conserved or that includes two or more sub-regions that are substantially conserved in a plurality of the genes in the alignment; and

preparing a series of primer sets, each primer set corresponding to one alignment and comprising a forward primer of from about 10 to about 40 bases complementary to a nucleotide segment of a first strand of the region and a reverse primer of from about 10 to about 40 bases complementary to a nucleotide segment of a second strand of the region;

wherein the forward and reverse primers corresponding to each alignment span a target region in each of the plurality of genes; and

wherein each primer set is effective to amplify the target regions from the plurality of genes when present in the sample by quantitative PCR.