# US 2014/0193823 A1 · Primer set and a method for identification of meat species

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0193823 A1
- Document ID: US20140193823A1
- Status: Published application
- Kind: A1
- Application number: 14/135,905
- Filed: 2013-12-20
- Published: 2014-07-10
- Earliest claimed: 2012-12-22
- Assignees: National Pingtung University of Science and Technology
- Applicants: National Pingtung University of Science and Technology
- Inventors: Shyang-Chwen Sheu; Ke-Wei Chen
- Web page: https://patentgrep.com/patent/US20140193823A1

## CPC

- C12Q 1/6888: for detection or identification of organisms
- 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

The invention discloses a primer set for identification of meat species. The primer set comprises a pair of outer primers used to amplify a sense strand of DNA fragment of mitochondrial cytochrome b between positions 51 and 507. The pair of outer primers comprises a forward outer primer and a backward outer primer. The primer set further comprises a pair of inner primers comprising a forward inner primer and a backward inner primer. The forward inner primer comprises a first annealing portion and a first warped portion. The backward inner primer comprises a second annealing portion and a second warped portion. Accordingly, the primer set is used to identify species of meat especially with DNA fragmentation due to processing processes of processed food.

## Claims

### Claim 1

A primer set for identification of meat species comprises:
a pair of outer primers used to amplify a sense strand of target fragment of mitochondrial cytochrome b between positions 51 and 507, comprising a forward outer primer and a backward outer primer, wherein the forward outer primer corresponds to a sense strand of a DNA fragment of mitochondrial cytochrome b between positions 51 and 325, wherein the backward outer primer corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b between positions 235 and 507; and
a pair of inner primers comprising a forward inner primer and a backward inner primer, wherein the forward inner primer comprises a first annealing portion and a first warped portion, wherein the first annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b between positions 74 and 360, wherein the first warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b between positions 116 and 401, the backward inner primer comprises a second annealing portion and a second warped portion, wherein the second annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b between positions 153 and 442, wherein the second warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b between positions 116 and 401.

### Claim 2 (depends on 1)

The primer set for identification of meat species as claimed in claim 1, with lengths of the forward outer primer and the backward outer primer being 18 to 23 bases.

### Claim 3 (depends on 1)

The primer set for identification of meat species as claimed in claim 1, with lengths of the first annealing portion, the first warped portion, the second annealing portion and the second warped portion being 20 to 25 bases.

### Claim 4 (depends on 1)

The primer set for identification of meat species as claimed in claim 1, wherein the forward outer primer corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Bos indicus between positions 303 and 325, wherein the backward outer primer corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Bos indicus between positions 490 and 507, wherein the first annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Bos indicus between positions 336 and 360, wherein the first warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Bos indicus between positions 377 and 401, wherein the second annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Bos indicus between positions 423 and 442, wherein the second warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Bos indicus between positions 464 and 483.

### Claim 5 (depends on 4)

The primer set for identification of meat species as claimed in claim 4, with the forward outer primer having a sequence set forth in SEQ ID NO: 1, with the backward outer primer having a sequence set forth in SEQ ID NO: 2, with the forward inner primer having a sequence set forth in SEQ ID NO: 3, with the backward inner primer having a sequence set forth in SEQ ID NO: 4.

### Claim 6 (depends on 1)

The primer set for identification of meat species as claimed in claim 1, wherein the forward outer primer corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Gallus gallus between positions 70 and 88, wherein the backward outer primer corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Gallus gallus between positions 241 and 259, wherein the first annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Gallus gallus between positions 100 and 119, wherein the first warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Gallus gallus between positions 140 and 161, wherein the second annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Gallus gallus between positions 168 and 189, wherein the second warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Gallus gallus between positions 215 and 234.

### Claim 7 (depends on 6)

The primer set for identification of meat species as claimed in claim 6, with the forward outer primer having a sequence set forth in SEQ ID NO: 5, with the backward outer primer having a sequence set forth in SEQ ID NO: 6, with the forward inner primer having a sequence set forth in SEQ ID NO: 7, with the backward inner primer having a sequence set forth in SEQ ID NO: 8.

### Claim 8 (depends on 1)

The primer set for identification of meat species as claimed in claim 1, wherein the forward outer primer corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Sus scrofa domestica between positions 51 and 68, wherein the backward outer primer corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Sus scrofa domestica between positions 235 and 255, wherein the first annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Sus scrofa domestica between positions 74 and 97, wherein the first warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Sus scrofa domestica between positions 116 and 140, wherein the second annealing portion corresponds to a sense strand of the DNA fragment of mitochondrial cytochrome b of Sus scrofa domestica between positions 153 and 177, wherein the second warped portion corresponds to an antisense strand of the DNA fragment of mitochondrial cytochrome b of Sus scrofa domestica between positions 210 and 229.

### Claim 9 (depends on 8)

The primer set for identification of meat species as claimed in claim 8, with the forward outer primer having a sequence set forth in SEQ ID NO: 9, with the backward outer primer having a sequence set forth in SEQ ID NO: 10, with the forward inner primer having a sequence set forth in SEQ ID NO: 11, with the backward inner primer having a sequence set forth in SEQ ID NO: 12.

### Claim 10 (depends on 1)

A method for identification of meat species comprising:
providing a primer set as claimed in claim 1;
mixing DNA template, a reaction buffer, dNTPs, the primer set and a Bst DNA polymerase to obtain a reaction mixture;
amplifying the target fragment of mitochondrial cytochrome b of the DNA template of the reaction mixture at 65° C. to obtain an amplified target fragment; and
analyzing the amplified target fragment by agarose electrophoresis.

### Claim 11 (depends on 10)

The method for identification of meat species as claimed in claim 10, with the forward outer primer having a sequence set forth in SEQ ID NO: 1, with the backward outer primer having a sequence set forth in SEQ ID NO: 2, with the forward inner primer having a sequence set forth in SEQ ID NO: 3, with the backward inner primer having a sequence set forth in SEQ ID NO: 4.

### Claim 12 (depends on 10)

The method for identification of meat species as claimed in claim 10, with the forward outer primer having a sequence set forth in SEQ ID NO: 5, with the backward outer primer having a sequence set forth in SEQ ID NO: 6, with the forward inner primer having a sequence set forth in SEQ ID NO: 7, with the backward inner primer having a sequence set forth in SEQ ID NO: 8.

### Claim 13 (depends on 10)

The method for identification of meat species as claimed in claim 10, with the forward outer primer having a sequence set forth in SEQ ID NO: 9, with the backward outer primer having a sequence set forth in SEQ ID NO: 10, with the forward inner primer having a sequence set forth in SEQ ID NO: 11, with the backward inner primer having a sequence set forth in SEQ ID NO: 12.

### Claim 14 (depends on 10)

The method for identification of meat species as claimed in claim 10, wherein the target fragment is amplified at 65° C. for 1 hour.

### Claim 15 (depends on 10)

The method for identification of meat species as claimed in claim 10, with the DNA template having amounts set forth in 1 to 100 ng.

### Claim 16 (depends on 10)

The method for identification of meat species as claimed in claim 10, wherein the primer set comprises the pair of outer primers being 0.5 μM and the pair of inner primers being 4 μM.