Detection of fermentation-related microorganisms

• US 6,248,519 B1
• Filed: 03/11/1998
• Issued: 06/19/2001
• Est. Priority Date: 03/11/1998
• Status: Expired due to Fees

• Current Assignee: E & J Gallo Winery
• Original Assignee: E & J Gallo Winery
• Inventors: Morenzoni, Richard A., Engel, Stacia R., Irelan, Nancy A., Descenzo, Richard A.
• Primary Examiner(s): Arthur, Lisa B.
• Assistant Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US09/037,990
• Time in Patent Office: 1,196 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 536/23.1, 536/24.32
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

DNA sequences are provided which are useful in identifying different fermentation-related microorganisms, such as those involved in fermentations. These DNA sequences can be used to provide oligonucleotide primers in PCR based analysis for the identification of fermentation-related microorganisms. The DNA sequences of the present invention include the internal transcribed spacer (ITS) of the ribosomal RNA gene regions of particular fermentation-related microorganisms, as well as oligonucleotide primers which are derived from these regions which are capable of identifying the particular microorganism.

Claims

1. An isolated double stranded nucleic acid which consists of a member selected from the group consisting of SEQ ID NOS:

13 to 20 and 25 to 34 and its complementary sequence.

2. An isolated nucleic acid which specifically hybridizes with a nucleic acid selected from the group consisting of SEQ ID NOS:

15 to 20 and 25 to 34, and their complementary sequences.

3. An oligonucleotide sequence for identification of a fermentation-related microorganism, wherein said sequence is selected from the group consisting of SEQ ID NOS:

37 to 64.

4. An oligonucleotide primer which is a fragment of the sequences according to claim 3, and which specifically hybridizes to the ITS1 or ITS2 of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomycodes ludwigii, Dekkera bruxellensis, Dekkera intermedia, Botrytis cinerea, Penicillium crustosum, Penicillium expansum, Hanseniaspora guilliermondii, Debaryomyces carsonii, Pichia anomala, Pichia kluyveri or Candida krusei.

5. An oligonucleotide primer for identification of a fermentation-related microorganism, wherein said primer specifically amplifies at least a portion of the ITS1 region of SEQ ID NOS:

13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 or at least a portion of the ITS2 region of SEQ ID NOS;

14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36, or which specifically amplifies at least a portion of the ITS region of a fermentation-related microorganism selected from the group consisting of Saccharomyces sp., Saccharomycodes sp., Debkera sp., Botrytis sp., Penicillium sp., Hanseniaspora sp., Debaryomyces sp., Pichia sp. and Candida sp., wherein said primer is selected from the group consisting of primers which contain at least 10 contiguous bases from one of SEQ ID NOS;

65 to 98, primers which contain which contain at least 10 contiguous bases from one of SEQ ID NOS;

65 to 98 contiguous with 1 to 15 flanking nucleotide bases in the 5′ and

/or 3′

direction of SEQ ID NOS;

37 to 64, and primers of 10 nucleotide bases or longer which contain at least 5 contiguous nucleotide bases from one of SEQ ID NOS;

65 to 98 contiguous with from 1 to 15 flanking nucleotide bases in the 5′ and

/or 3′

direction of SEQ ID NOS;

37 to 64 wherein said primer specifically amplifies at least a portion of the ITS region of members of a specific species of fermentation-related microorganism but does not amplify the ITS regions of other species of fermentation-related microorganisms.

44. A kit comprising a carrier to receive therein one or more containers, at least one of said containers comprising an oligonucleotide primer according to claim 5.

6. A pair of oligonucleotide primers for use in the amplification-based detection of an internal transcribed spacer sequence of a fermentation-related microorganism, wherein said primers specifically amplify at least a portion of the ITS1 region of SEQ ID NOS:

13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 or at least a portion of the ITS2 region of SEQ ID NOS;

14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36, or which specifically amplify at least a portion of the ITS region of a fermentation-related microorganism selected from the group consisting of Saccharomyces sp., Saccharomycodes sp., Dekkera sp., Botrytis sp., Penicillium sp., Hanseniaspora sp., Debaryomyces sp., Pichia sp. and Candida sp., wherein said primers are selected from the group consisting of primers which contain at least 10 contiguous nucleotide bases from one of SEQ ID NOS;

65 to 98, primers which contain at least 10 contiguous nucleotide bases from one of SEQ ID NOS;

65 to 98 contiguous with 1 to 15 flanking nucleotide bases in the 5′ and

/or 3′

direction of SEQ ID NOS;

37 to 64, and primers of 10 bases or longer which contain at least 5 contiguous nucleotide bases from one of SEQ ID NOS;

65 to 98 contiguous with 1 to 15 nucleotide flanking bases in the 5′ and

/or 3′

direction of SEQ ID NOS;

37 to 64 wherein said primers specifically amplify at least a portion of the ITS region of members of a specific species of fermentation-related microorganism but do not amplify the ITS regions of other species of fermentation-related microorganisms.

7. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

65 and SEQ ID NO;

66.

8. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

67 and SEQ ID NO;

68.

9. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

69 and SEQ ID NO;

70.

10. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

71 and SEQ ID NO;

72.

11. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

73 and SEQ ID NO;

74.

12. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

75 and SEQ ID NO;

78.

13. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

76 and SEQ ID NO;

79.

14. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

77 and SEQ ID NO;

80.

15. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

81 and SEQ ID NO;

82.

16. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

82 and SEQ ID NO;

83.

17. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

84 and SEQ ID NO;

85.

18. The pair oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

86 and SEQ ID NO;

87.

19. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

86 and SEQ ID NO;

88.

20. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

89 and SEQ ID NO;

91.

21. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

90 and SEQ ID NO;

91.

22. The pair oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

94 and SEQ ID NO;

95.

23. The pair of oligonucleotide primers according to claim 6 which comprises SEQ ID NO:

97 and SEQ ID NO;

98.

24. A method for detection of a fermentation-related microorganism comprising:

(a) obtaining DNA from a fungal culture or colony isolated from a fermentation, or from an organism present in a fermentation beverage;

(b) amplifying a part of the internal transcribed spacer sequence of said fermentation-related microorganism using said DNA as a template in a polymerase chain reaction with a pair of oligonucleotide primers according to claim 6; and

(c) visualizing said amplified part of the internal transcribed spacer sequence to determine whether said fermentation-related microorganism is present.

25. The method according to claim 24, wherein said fermentation-related microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomycodes ludwigii, Dekkera bruxellensis, Dekkera intermedia, Botrytis cinerea, Penicillium crustosum, Penicillium expansum, Hanseniaspora guilliermondii, Debaryomyces carsonii, Pichia anomala, Pichia kluyveri, and Candida krusei.

26. The method according to claim 24, wherein said fungal culture is isolated from a wine fermentation or said fermentation beverage is a wine fermentation beverage.

27. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

65 and SEQ ID NO;

66.

28. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

67 and SEQ ID NO;

68.

29. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

69 and SEQ ID NO;

70.

30. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

71 and SEQ ID NO;

72.

31. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

73 and SEQ ID NO;

74.

32. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

75 and SEQ ID NO;

78.

33. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

76 and SEQ ID NO;

79.

34. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

77 and SEQ ID NO;

80.

35. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

81 and SEQ ID NO;

82.

36. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

82 and SEQ ID NO;

83.

37. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

84 and SEQ ID NO;

85.

38. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

86 and SEQ ID NO;

87.

39. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

86 and SEQ ID NO;

88.

40. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

89 and SEQ ID NO;

91.

41. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

90 and SEQ ID NO;

91.

42. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

94 and SEQ ID NO;

95.

43. The method according to claim 24, wherein the pair of oligonucleotide primers comprises SEQ ID NO:

97 and SEQ ID NO;

98.

45. A kit comprising a carrier to receive therein one or more containers, at least one of said containers comprising a pair of oligonucleotide primers according to claim 6.