# US 5,348,857 A · Probes and method for identifying species and biovars of brucella

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- Number: US 5,348,857 A
- Document ID: US5348857A
- Status: Granted
- Kind: A
- Application number: 07/972,791
- Filed: 1992-11-06
- Granted: 1994-09-20
- Earliest claimed: 1990-05-22
- Assignees: Texas A&M University
- Inventors: Thomas A. Ficht; L. Garry Adams
- Web page: https://patentgrep.com/patent/US5348857A

## CPC

- C12Q 1/689: for bacteria
- Y10S 436/811: Test for named disease, body condition or organ function
- 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
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

A method for detecting Brucella infection in an animal which is reliable, rapid, and able to identify species and biovars of Brucella. The detection method includes the amplification of the omp2 gene locus of Brucella and analysis of restriction digestion fragments specific to Brucella and to individual species and groups of biovars of Brucella.

## Claims

### Claim 1

A method for identifying a species or biovar of Brucella comprising the steps of:
releasing DNA from a test sample;
amplifying a gene sequence of a Brucella omp2 gene locus as defined in FIGS. 2A-O from the released DNA, said gene sequence having sufficient diversity to distinguish species of Brucella;
analyzing the hybridization of the amplified gene sequence with a panel of DNA probes to identify a species or biovar of Brucella.

### Claim 2 (depends on 1)

The method of claim 1 wherein said amplified gene sequence is a sequence between nucleotides 2470 and 3346 of the Brucella omp2 gene consensus sequence.

### Claim 3 (depends on 1)

The method of claim 1 wherein said amplifying is by polymerase chain reaction.

### Claim 4 (depends on 3)

The method of claim 3, wherein the polymerase chain reaction is primed with an oligonucleotide pair which anneals to the omp2 gene locus of Brucella to amplify a sequence between nucleotides 2470 and 3346 of the Brucella omp2 consensus sequence.

### Claim 5 (depends on 3)

The method of claim 3, wherein the polymerase chain reaction is primed with an oligonucleotide pair selected from those having Seq. Id. Nos. 9-20.

### Claim 6 (depends on 4)

The method of claim 4, wherein said oligonucleotide pair is that having Seq. Id. Nos. 19 and 20.

### Claim 7 (depends on 6)

The method of claim 6, wherein said panel of DNA probes includes one or more of the probes having Seq. Id. Nos. 21-32.

### Claim 8 (depends on 7)

The method of claim 7, wherein said panel includes at least two probes having Seq. Id. Nos. 21-32.

### Claim 9 (depends on 1)

The method of claim 1, wherein said test sample is animal fluid or tissue.

### Claim 10 (depends on 7)

The method of claim 7 wherein said test sample is urine, blood, milk, semen, vaginal or rectal secretions.

### Claim 11 (depends on 8)

The method of claim 8, wherein said test sample is milk.

### Claim 12 (depends on 1)

The method of claim 1, wherein said analyzing is by dot blot, DNA hybridization, or by agarose gel electrophoresis.

### Claim 13 (depends on 12)

The method of claim 12, wherein said analyzing is using radiolabeled oligonucleotide probes.