# US 5,705,339 A · Methods for the detection of the bacterial agents causing spoilage of beer

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- Number: US 5,705,339 A
- Document ID: US5705339A
- Status: Granted
- Kind: A
- Application number: 08/455,063
- Filed: 1995-05-31
- Granted: 1998-01-06
- Earliest claimed: 1993-09-10
- Assignees: Amoco Corporation
- Inventors: Raymond M. Nietupski; Benjamin B. Stone; William G. Weisburg
- Web page: https://patentgrep.com/patent/US5705339A

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12Q 1/689: for bacteria
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Nucleic acid sequences which preferentially bind to the rRNA or rDNA of microorganisms which cause the spoilage of beer are disclosed. The beer spoilage microorganisms are predominantly of the genera Lactobacillus and Pediococcus. The nucleic acids may be used as probes in assays to detect the presence of these microorganisms. Kits containing two or more probes are also described.

## Claims

### Claim 1

A method of detecting Pediococcus damnosus in a sample, said method comprising the steps of
contacting the sample under stringent hybridization conditions with an isolated nucleic acid probe fully complementary to probe 2858 (SEQ ID NO:1), probe 2861 (SEQ ID NO:2), or probe 2867 (SEQ ID NO:3), or the complement of any one of probes 2858, 2861, or 2867, to form a detectable complex with 16S rRNA or rDNA of Pediococcus damnosus in the sample, if any, under said stringent hybridization conditions, wherein said probe does not form a detectable complex with rRNA or rDNA of non-Pediococcus damnosus bacteria under said stringent hybridization conditions; and
detecting the complexes as an indication of Pediococcus damnosus in the sample.

### Claim 2 (depends on 1)

A method of claim 1, wherein said probe is probe 2858 (SEQ ID NO:1) or the complement of probe 2858.

### Claim 3 (depends on 1)

A method of claim 1, wherein said probe comprises one or more of a detection ligand, an end cap, or a homopolymeric nucleotide sequence.

### Claim 4 (depends on 1)

A method of claim 1, wherein said probe comprises an amplifiable Q-beta midivariant nucleotide sequence.

### Claim 5

A method of detecting Lactobacillus brevis in a sample, said method comprising the steps of
contacting the sample under stringent hybridization conditions with an isolated nucleic acid probe fully complementary to probe 2868 (SEQ ID NO:6), 2869 (SEQ ID NO:7), 2880 (SEQ ID NO:8), 2891 (SEQ ID NO:9), 2892 (SEQ ID NO:10), or 2895 (SEQ ID NO:11), or the complement of any one of probes 2868, 2869, 2880, 2891, 2892, or 2895, to form a detectable complex with 16S or 23S rRNA or rDNA of Lactobacillus brevis in the sample, if any, under said stringent hybridization conditions, wherein said probe does not form a detectable complex with rRNA or rDNA of non-Lactobacillus brevis bacteria under said stringent hybridization conditions; and
detecting the complexes as an indication of Lactobacillus brevis in the sample.

### Claim 6 (depends on 5)

A method of claim 5, wherein said probe is selected from the group consisting of probe 2869 (SEQ ID NO:7) or the complement of probe 2869.

### Claim 7 (depends on 5)

A method of claim 5, wherein said probe comprises one or more of a detection ligand, an end cap, or a homopolymeric nucleotide sequence.

### Claim 8 (depends on 5)

A method of claim 5, wherein said probe comprises an amplifiable Q-beta midivariant nucleotide sequence.

### Claim 9

A method of detecting Lactobacillus or Pediococcus bacteria that cause beer spoilage in a sample of beer, said method comprising the steps of
contacting the sample under stringent hybridization conditions with an isolated nucleic acid probe fully complementary to probe 2876 (SEQ ID NO:4), 2877 (SEQ ID NO:5), 2899 (SEQ ID NO:12), 2904 (SEQ ID NO:13), 2896 (SEQ ID NO:14), 2873 (SEQ ID NO:15), 2887 (SEQ ID NO:17), 2875 (SEQ ID NO:18), or 2901 (SEQ ID NO:19), or the complement of any one of probes 2876, 2877, 2899, 2904, 2896, 2873, 2887, 2875, and 2901, to form a detectable complex with 16S or 23S rRNA or rDNA of Lactobacillus or Pediococcus in the sample, if any, under said stringent hybridization conditions, wherein said probe does not form a detectable complex with rRNA or rDNA of non-Lactobacillus or non-Pediococcus bacteria under said stringent hybridization conditions; and
detecting the complexes as an indication of Lactobacillus or Pediococcus that cause beer spoilage in the sample.

### Claim 10 (depends on 9)

A method of claim 9, wherein said probe is probe 2873 (SEQ ID NO:15) or the complement of probe 2873.

### Claim 11 (depends on 9)

A method of claim 9, wherein said probe comprises one or more of a detection ligand, an end cap, or a homopolymeric nucleotide sequence.

### Claim 12 (depends on 9)

A method of claim 9, wherein said probe comprises an amplifiable Q-beta midivariant nucleotide sequence.

### Claim 13 (depends on 5)

A method of claim 5, wherein said probe is probe 2891 (SEQ ID NO:9) or the complement of probe 2891.

### Claim 14 (depends on 9)

A method of claim 9, wherein said probe is probe 2876 (SEQ ID NO:4) or the complement of probe 2876.

### Claim 15 (depends on 9)

A method of claim 9, wherein said probe is probe 2899 (SEQ ID NO:12) or the complement of probe 2899.

### Claim 16 (depends on 9)

A method of claim 9, wherein said probe is probe 2875 (SEQ ID NO:18) or the complement of probe 2875.