# US 5,705,332 A · Detection and identification of Salmonella and Shigella

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- Number: US 5,705,332 A
- Document ID: US5705332A
- Status: Granted
- Kind: A
- Application number: 08/232,778
- Filed: 1994-04-25
- Granted: 1998-01-06
- Earliest claimed: 1994-04-25
- Assignees: University of Hawaii
- Inventors: Bruce Roll
- Web page: https://patentgrep.com/patent/US5705332A

## CPC

- C12Q 1/689: for bacteria
- C12Q 2600/16: Primer sets for multiplex assays
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention is directed to methods and reagents for the specific detection and presumptive identification of various bacteria associated with waterborne infectious disease. In particular, this invention relates to methods and reagents for the specific detection and identification of Salmonella and Shigella in environmental samples such as water and sewage.

## Claims

### Claim 1

A method for amplification of nucleic acid from one or more enteric pathogens comprising the steps of:
a) providing a test sample suspected of containing amplifiable nucleic acid from said one or more enteric pathogens;
b) isolating said amplifiable nucleic acid from said test sample;
c) combining said amplifiable nucleic acid with amplification reagents, and at least two primers selected from the group consisting of primers having the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 to form a reaction mixture; and
d) combining said reaction mixture with an amplification enzyme under conditions wherein said amplifiable nucleic acid is amplified to form amplification product.

### Claim 2 (depends on 1)

The method of claim 1 further comprising the step of detecting said amplification product.

### Claim 3 (depends on 2)

The method of claim 2, in which said detecting is accomplished by hybridization of said amplification product with a probe having the nucleic acid sequence set forth in SEQ ID NO:5.

### Claim 4 (depends on 2)

The method of claim 2, in which said detecting is accomplished by hybridization of said amplification product with a probe selected from the group consisting of the nucleic acid sequences as set forth in SEQ ID NO:8 and SEQ ID NO:9.

### Claim 5 (depends on 1)

The method of claim 1, wherein said primers hybridize to nucleic acid from said enteric pathogen selected from the group consisting of Salmonella and Shigella species.

### Claim 6 (depends on 1)

The method of claim 1, wherein said test sample is selected from the group comprising sewage, sludge, soil, food, feed, and water.

### Claim 7

A method for amplification of nucleic acid from a plurality of enteric pathogens comprising the steps of:
a) providing a test sample suspected of containing amplifiable nucleic acid of said plurality of enteric pathogens;
b) isolating said amplifiable nucleic acid from said test sample;
c) combining said amplifiable nucleic acid with amplification reagents, and all of the primers set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7, to form a reaction mixture;
d) combining said reaction mixture with an amplification enzyme under conditions wherein said amplifiable nucleic acid is amplified to form amplification products; and
e) detecting said amplification products.

### Claim 8 (depends on 7)

The method of claim 7, wherein said detecting comprises hybridizing said amplification product with the probe having the nucleic acid sequences selected from the group consisting of SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:9.

### Claim 9 (depends on 1)

The method of claim 1, wherein said enteric pathogens consist of the Salmonella and Shigella species.

### Claim 10 (depends on 1)

The method of claim 1, wherein said test sample is selected from the group comprising sewage, sludge, soil, food, feed, and water.

### Claim 11

A method for amplification of nucleic acid from an enteric pathogen comprising the steps of:
a) providing a test sample suspected of containing amplifiable nucleic acid of said enteric pathogen;
b) isolating said amplifiable nucleic acid from said test sample;
c) combining said amplifiable nucleic acid with amplification reagents, and the primers set forth in SEQ ID NO:1 and SEQ ID NO:2, to form a first reaction mixture;
d) combining said reaction mixture with an amplification enzyme under conditions wherein said amplifiable nucleic acid is amplified to form a first amplification product;
e) combining said first reaction mixture with amplification reagents, and the primers set forth in SEQ ID NO:3, and SEQ ID NO:4, to form a second reaction mixture;
f) combining said second reaction mixture with an amplification enzyme under conditions wherein said amplifiable nucleic acid is amplified to form a second amplification product; and
g) detecting said first or second amplification product.

### Claim 12 (depends on 11)

The method of claim 11, wherein said detecting comprises hybridizing said amplification product with a probe having the nucleic acid sequence set forth in SEQ ID NO:5.

### Claim 13 (depends on 12)

The method of claim 12, wherein said enteric pathogen is Salmonella.

### Claim 14 (depends on 12)

The method of claim 12, wherein said test sample is selected from the group comprising sewage, sludge, soil, food, feed, and water.