# US 8,148,511 B2 · Methods and compositions for the detection and quantification of E. coli and Enterococcus

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- Number: US 8,148,511 B2
- Document ID: US8148511B2
- Status: Granted
- Kind: B2
- Application number: 11/859,032
- Filed: 2007-09-21
- Published: 2008-09-25
- Granted: 2012-04-03
- Earliest claimed: 2006-09-28
- Assignees: The University of North Carolina at Chapel Hill
- Inventors: Rachel T. Noble; Angelia D. Blackwood
- Web page: https://patentgrep.com/patent/US8148511B2

## CPC

- C12Q 1/689: for bacteria
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- 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 present invention is drawn to methods and compositions for the rapid assessment of fecal indicator bacteria in a sample. Provided herein are novel primer and probe compositions for use in detecting the presence of these organisms in a sample, particularly using quantitative PCR methods. Provided herein are novel oligonucleotide primers and probes, including the primers set forth in SEQ ID NO:1-4, the novel oligonucleotide probe sequences set forth in SEQ ID NO:5-8, and methods for using these primers and probes for the detection and/or quantification of fecal indicator bacteria, particularly E. coli and Enterococcus spp. in a sample.

## Claims

### Claim 1

A composition for the detection of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample, wherein said composition comprises, in the 5′ to 3′ direction, one of a fluorophore or a quencher molecule, an oligonucleotide probe comprising SEQ ID NO:5 or 7, the other of the fluorophore or quencher molecule, a PCR blocker moiety, and an oligonucleotide comprising SEQ ID NO:1, wherein a portion of the oligonucleotide probe SEQ ID NO:5 or 7 binds to an amplification product of SEQ ID NO:1.

### Claim 2

A composition for the detection of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample, wherein said composition comprises, in the 5′ to 3′ direction, one of a fluorophore or a quencher molecule, an oligonucleotide probe comprising SEQ ID NO:6 or 8, the other of the fluorophore or quencher molecule, a PCR blocker moiety, and an oligonucleotide comprising SEQ ID NO:3 wherein a portion of the oligonucleotide probe SEQ ID NO:6 or 8 binds to an amplification product of SEQ ID NO:3.

### Claim 3 (depends on 1)

The composition of claim 1, wherein said PCR blocker moiety is hexethylene glycol (HEG).

### Claim 4 (depends on 2)

The composition of claim 2, wherein said PCR blocker moiety is hexethylene glycol (HEG).

### Claim 5 (depends on 1)

The composition of claim 1 comprising, in the 5′ to 3′ direction, a fluorophore, an oligonucleotide comprising SEQ ID NO:7, a quencher, a PCR blocker moiety, and an oligonucleotide comprising SEQ ID NO:1.

### Claim 6 (depends on 2)

The composition of claim 2 comprising, in the 5′ to 3′ direction, a fluorophore, an oligonucleotide comprising SEQ ID NO:8, a quencher, a PCR blocker moiety, and an oligonucleotide comprising SEQ ID NO:3.

### Claim 7 (depends on 1)

A kit for the PCR detection of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample comprising the composition of claim 1 and an oligonucleotide comprising SEQ ID NO:2.

### Claim 8 (depends on 2)

A kit for the PCR detection of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample comprising the composition of claim 2 and an oligonucleotide comprising SEQ ID NO:4.

### Claim 9 (depends on 1)

A kit for the PCR detection of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample comprising the composition of claim 1.

### Claim 10 (depends on 2)

A kit for the PCR detection of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample comprising the composition of claim 2.

### Claim 11

A method for detecting the presence of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample using polymerase-based amplification of a target nucleic acid region present in said fecal indicator bacteria, said method comprising:
a) providing a test environmental sample suspected of containing fecal indicator bacteria;
b) contacting said environmental sample with at least a first and a second oligonucleotide primer under conditions sufficient to provide polymerase-based nucleic acid amplification products comprising the target region, wherein said at least a first and a second oligonucleotide primer is selected from the group consisting of:
i) a first oligonucleotide primer comprising SEQ ID NO:1 and a second oligonucleotide primer comprising SEQ ID NO:2; or,
ii) a first oligonucleotide primer comprising SEQ ID NO:3 and a second oligonucleotide primer comprising SEQ ID NO:4; and,
c) detecting the amplified products.

### Claim 12 (depends on 11)

The method of claim 11, wherein said first oligonucleotide primer comprising SEQ ID NO:1 further comprises a probe covalently attached to the 5′ end of the primer, wherein said probe comprises, in the 5′ to 3′ direction, one of a fluorophore or a quencher molecule, an oligonucleotide probe comprising SEQ ID NO:5 or 7, the other of the fluorophore or the quencher molecule, and a PCR blocker moiety, wherein nucleotides 1-11 of SEQ ID NO:5 or nucleotides 3-13 of SEQ ID NO:7 bind to an amplification product of SEQ ID NO:1 and SEQ ID NO: 2.

### Claim 13 (depends on 11)

The method of claim 11, wherein said first oligonucleotide primer comprising SEQ ID NO:3 further comprises a probe covalently attached to the 5′ end of the primer, wherein said probe comprises, in the 5′ to 3′ direction, one of a fluorophore or a quencher molecule, an oligonucleotide probe comprising SEQ ID NO:6 or 8, the other of the fluorophore or the quencher molecule, and a PCR blocker moiety wherein nucleotides 1-21 of SEQ ID NO:5 or nucleotides 5-25 of SEQ ID NO:8 bind to an amplification product of SEQ ID NO:3 and SEQ ID NO:4.

### Claim 14 (depends on 11)

The method of claim 11, wherein said method further comprises the step of isolation of bacterial nucleic acid from the sample provided in step (a).

### Claim 15 (depends on 14)

The method of claim 14, wherein said method of isolation comprises bead beating.

### Claim 16 (depends on 12)

The method of claim 12, wherein said PCR blocker moiety is hexethylene glycol.

### Claim 17 (depends on 11)

The method of claim 11, wherein said polymerase-based amplification is quantitative polymerase chain reaction (QPCR).

### Claim 18

A method for detecting the presence of a multiplicity of strains and species of fecal indicator bacteria in an environmental sample using polymerase-based amplification of a target nucleic acid region present in said fecal indicator bacteria, said method comprising:
a) providing a test environmental sample suspected of containing fecal indicator bacteria;
b) contacting said environmental sample with at least a first and a second oligonucleotide primer under conditions sufficient to provide polymerase-based nucleic acid amplification products comprising the target region, wherein said at least a first and a second oligonucleotide primer is selected from the group consisting of:
i) a first oligonucleotide primer comprising at least 15 nucleotides of SEQ ID NO:1 and a second oligonucleotide primer comprising at least 15 nucleotides of SEQ ID NO:2; or,
ii) a first oligonucleotide primer comprising at least 15 nucleotides SEQ ID NO:3 and a second oligonucleotide primer comprising at least 15 nucleotides of SEQ ID NO:4; and,
c) detecting the amplified products.