Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-11
2001-06-19
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300, C536S024320
Reexamination Certificate
active
06248519
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to assays to detect fermentation-related microorganisms.
DESCRIPTION OF THE RELATED ART
In traditional wine-making, the indigenous yeasts ferment the grape must. Most modern wine-makers, however, inoculate with a pure culture of a selected yeast strain to ensure a rapid, reliable and predictable fermentation. It is thought that indigenous yeasts are suppressed by the competitive effect of addition of a high-density monoculture, but some evidence suggests that indigenous yeast can still participate in the fermentation. A range of commercial yeasts with different wine-making characteristics is available, and a number of those yeast strains may be used in a single winery. Furthermore, unwanted microorganisms may be present which lead to spoilage. Therefore, there is a need for a rapid, simple and accurate method for identifying microorganisms in starter cultures and fermentations.
Past techniques for detecting and identifying fermentation-related microorganisms, especially yeast, include colony morphology, fermentation performance, sugar fermentation tests, tolerance to various stresses (e.g., ethanol tolerance in brewing), phenotypes with functional relevance (e.g., flocculation in brewing), nutritional requirements (e.g., oxygen), and resistance and sensitivity levels of cycloheximide. These methods, however, have numerous disadvantages, including lengthy analysis periods, inability to differentiate, e.g., different strains of yeast, and lack of reproducibility.
Recent developments in molecular biology and protein chemistry have provided new methods for identifying microorganisms, including DNA restriction fragment length polymorphisms, protein electrophoresis patterns and chromosome fingerprinting. Such techniques have been used for identifying fermentation-related microorganisms. See, for example, Casey et al,
Journal of the American Society of Brewing Chemists,
48(3):100-106, 1990; Degre et al,
American Journal of Enology and Viticulture,
40(4)309-315, 1989; Guillamon et al,
Systematic and Applied Microbiology,
19:122-132, 1992; Hoeben et al,
Current Genetics,
10:371-379, 1986; Mozina et al,
Letters in Applied Microbiology,
24(4):311-315, 1997; Paffetti et al,
Research Microbiology,
146:587-594, 1995; Panchal et al,
Journal of the Institute of Brewing,
93:325-327, 1987; Querol et al,
Systematic and Applied Microbiology,
15:439-446, 1992; Vezinhet et al,
Applied Microbiology and Biotechnology,
32:568-571, 1990; and Vezinhet et al,
American Journal of Enology and Viticulture,
43(1):83-86, 1992.
Polymerase chain reaction (PCR)-based techniques have also been used to detect fermentation-related microorganisms. See, for example, DeBarros Lopes et al,
Applied and Environmental Microbiology,
62(12):4514-4520, 1996; Fell,
Molecular Marine Biology and Biotechnology,
2(3)174-180, 1993; Fell,
Journal of Industrial Microbiology,
14(6):475-477, 1995; Ibeas et al,
Applied and Environmental Microbiology,
62(3):998-1003, 1996; Lavallee et al,
American Journal of Enology and Viticulture,
45(1):86-91, 1994; Lieckfeldt et al,
Journal of Basic Microbiology,
33(6)413-425, 1993; and Ness et al,
J. Sci. Food Agric.,
62:89-94, 1993.
Ribosomal genes are suitable for use as molecular probe targets because of their high copy number. Non-transcribed and transcribed spacer sequences associated with ribosomal genes are usually poorly conserved and, thus, are advantageously used as target sequences for the detection of recent evolutionary divergence. Fungal rRNA genes are organized in units. Each unit encodes mature subunits of 18S, 5.8S, and 28S rRNA. The internal transcribed spacer (ITS) region lies between the 18S and 28S rRNA genes and contains two variable non-coding spacers (referred to as ITS1 and ITS2) and the 5.8S rRNA gene (White et al., 1990; In:
PCR Protocols;
Eds.: Innes et al.; pages 315-322). In addition, the transcriptional units are separated by non-transcribed spacer sequences (NTSs). The ITS and NTS sequences are particularly suitable for the detection of different fungal pathogens.
Kumeda et al (
Applied and Environmental Microbiology,
62(8):2947-2952, 1996) describes use of PCR to amplify ribosomal DNA internal transcribed spacers in order to differentiate species of Aspergillus Section Flavi. The ITS1-5.8S-ITS2 region was amplified using universal primers, and the PCR product analyzed by the principle of single-strand conformation polymorphism. In addition, Gardes et al (in:
Methods in Molecular Biology, Vol.
50:
Species Diagnostics Protocols: PCR and Other Nucleic Acid Methods,
Ed. J. P. Clapp, Humana Press, Totowa, N.J., (1996) pp. 177-186) describes restriction fragment length polymorphism (RFLP) analysis of fungal ITS regions amplified by PCR.
The PCR amplification of fungal ITS has also been described using other than universal primers. These methods allow for more specificity in identifying classes of fungi, or particular species of fungi. Thus, Gardes and Bruns (
Molecular Ecology,
2:113-118, 1993) identified ITS primers which allow differentiation of DNA from basidiomycetes against ascomycete DNA. Identification of specific species has been observed using PCR primers directed to unique sequences in the ITS1 and/or ITS2 regions of fungal pathogens. See, for example, Hamelin et al,
Applied and Environmental Microbiology,
62(11):4026-4031, 1996; Mazzola et al,
Phytopathology,
86(4):354-360, 1996; O'Gorman et al,
Canadian Journal of Botany,
72:342-346, 1994; and U.S. Pat. No. 5,585,238 to Ligon et al.
The present invention addresses the problem of detecting and identifying fermentation-related microorganisms by PCR-based techniques.
SUMMARY OF THE INVENTION
The present invention is directed to the identification of different fermentation-related microorganisms, particularly those involved in the production of wine. The present invention provides DNA sequences which exhibit variability between different fermentation-related microorganisms. In particular, the present invention identifies regions of DNA sequence located in the internal transcribed spacer (ITS) of the ribosomal RNA gene regions of various fermentation-related microorganisms. Primers derived from the ITS can be used in polymerase chain reaction (PCR) based diagnostic assays to determine the presence or absence of specific fermentation-related microorganisms, including those involved in the production of wine. The primers can also be used as molecular probes to detect the presence of target DNA.
Thus, in one aspect, the present invention provides an isolated double stranded nucleic acid of the full length ITS1 or ITS2 region of a fermentation-related microorganism. More particularly, the DNA sequence is selected from among Sequence ID NOS: 13 to 36.
In another aspect, the present invention provides an oligonucleotide primer for identification of a fermentation-related microorganism, wherein the primer is a divergent portion of the ITS1 or ITS2 region of a fermentation-related microorganism. More particularly, the oligonucleotide primer is selected from among Sequence ID NOS: 65 to 98. Furthermore, the oligonucleotide primers may be selected from among sequences 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 nucleotide bases in the 5′ and/or 3′ direction of corresponding 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 nucleotide bases in the 5′ and/or 3′ direction of corresponding SEQ ID NOS: 37 to 64. A pair of the foregoing oligonucleotide primers for use in the amplification-based detection of an ITS of a fermentation-related microorganism is also provided.
In yet another aspect, a method is provided for detection of a fermentation-related microorganism which comprises: (a) obtaining DNA from a funga
Descenzo Richard A.
Engel Stacia R.
Irelan Nancy A.
Morenzoni Richard A.
Arthur Lisa B.
E & J Gallo Winery
Goldberg Jeanine
Marshall O'Toole Gerstein Murray & Borun
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