# US 9,040,242 B2 · Method to amplify nucleic acids to generate fluorescence labeled fragments of conserved and arbitrary products

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- Number: US 9,040,242 B2
- Document ID: US9040242B2
- Status: Granted
- Kind: B2
- Application number: 13/553,256
- Filed: 2012-07-19
- Published: 2013-01-31
- Granted: 2015-05-26
- Earliest claimed: 2011-07-25
- Assignees: E. I. DuPont de Nemours and Company
- Applicants: Anjana Agarwal; Mark A. Jensen
- Inventors: Anjana Agarwal; Mark A. Jensen
- Web page: https://patentgrep.com/patent/US9040242B2

## CPC

- C12Q 1/689: for bacteria
- C12Q 1/6846: {Common amplification features}
- C12Q 1/686: Polymerase chain reaction [PCR]
- 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

Disclosed herein are methods for the identification of the species, serotype, and strain of a microorganism. Also disclosed are primers for use in detecting such microorganisms and kits comprising such primers.

## Claims

### Claim 1

A method for the identification of the species, serotype, and strain of a microorganism comprising:
(a) amplifying DNA comprising variable sequences interspersed between highly conserved rDNA sequences by PCR and amplifying additional genomic sequences by random amplified polymorphic DNA (RAPD) PCR using a first primer of 13-14 bases in length and a second primer of 11-13 bases in length, said first primer comprising:
(i) 11-12 contiguous bases from a highly conserved 16S rDNA region;
(ii) 2 bases at the 5′ end of said first primer that are not complementary to said highly conserved 16S rDNA region; and
(iii) a fluorescent label; and
(b) separating the amplified DNA produced in step (a).

### Claim 2 (depends on 1)

The method of claim 1, wherein said first primer is a forward primer.

### Claim 3 (depends on 2)

The method of claim 2, wherein said first primer comprises SEQ ID NO:1.

### Claim 4 (depends on 3)

The method of claim 3, wherein said first primer is SEQ ID NO:2.

### Claim 5 (depends on 1)

The method of claim 1, wherein said second primer comprises 11-13 contiguous bases from 23S rDNA.

### Claim 6 (depends on 5)

The method of claim 5, wherein said second primer is SEQ ID NO:3 or SEQ ID NO:4.

### Claim 7 (depends on 1)

The method of claim 1, wherein said amplifying step utilizes a third primer of 11-13 bases in length, said third primer comprising 11-13 contiguous bases from 23S rDNA, wherein said third primer is a different length than said second primer, and wherein one of either the second primer or third primer comprises 13 contiguous bases from 23S rDNA.

### Claim 8 (depends on 1)

The method of claim 1, wherein step (b) is accomplished by capillary electrophoresis.