Methods and oligonucleotides for the detection of Salmonella sp., E. coli O157:H7, and Listeria monocytogenes

• US 20030022214A1
• Filed: 06/21/2002
• Published: 01/30/2003
• Est. Priority Date: 06/22/2001
• Status: Active Grant

• Current Assignee: Marshfield Clinic
• Original Assignee: Marshfield Clinic
• Inventors: Vevea, Dirk N., Ellingson, Jay L.E.
• Granted Patent: US 7,241,566 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A method for detecting a Salmonella species, E. coli O157:H7, or Listeria monocytogenes is disclosed. The method involves amplifying a genomic nucleotide sequence of a corresponding species and detecting the amplification product. Various primers and probes that can be used in the method are also disclosed. In one embodiment, the amplification step of the method is accomplished by real-time PCR and the amplification product is detected by fluorescence resonance energy transfer using a pair of labeled polynucleotides.

Claims

1. An isolated nucleic acid comprising at least 12 consecutive nucleotides of a nucleotide sequence selected from SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

9, and SEQ ID NO;

10.

2. The isolated nucleic acid of claim 1, wherein the nucleic acid comprises at least 15 consecutive nucleotides of the nucleotide sequence.

3. The isolated nucleic acid of claim 1, wherein the nucleic acid comprises at least 18 consecutive nucleotides of the nucleotide sequence.

4. The isolated nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence selected from SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

9, and SEQ ID NO;

10.

5. A pair of labeled nucleic acids:

the first labeled nucleic acid comprises at least 12 consecutive nucleotides of a nucleotide sequence selected from SEQ ID NO;

13, the complement of SEQ ID NO;

13, SEQ ID NO;

14, the complement of SEQ ID NO;

14, SEQ ID NO;

15, and the complement of SEQ ID NO;

15 on the 3′

end region, and labeling molecule one at the 3′

end; and

the second labeled nucleic acid comprises at least 12 consecutive nucleotides of the same nucleotide sequence on the 5′

end region, and labeling molecule two at the 5′

end;

wherein when the two labeled nucleic acids hybridize to the complement of the nucleotide sequence, the nucleotide at the 3′

end of the first labeled nucleic acid and the nucleotide at the 5′

end of the second labeled nucleic acid are separated by less than six nucleotides and at least one of labeling molecule one and labeling molecule two changes emission intensity at a wavelength due to energy transfer between the two labeling molecules.

6. The pair of labeled nucleic acids of claim 5, wherein the first labeled nucleic acid comprises at least 15 consecutive nucleotides of the nucleotide sequence and the second labeled nucleic acid comprises at least 15 consecutive nucleotides of the same nucleotide sequence.

7. The pair of labeled nucleic acids of claim 5, wherein labeling molecule one is fluorescein, and labeling molecule two is selected from LC RED 640 and LC RED 705.

8. The pair of labeled nucleic acids of claim 5, wherein the nucleic acid sequences of each pair are selected from SEQ ID NO:

3 and SEQ ID NO;

4, the complement of SEQ ID NO;

3 and the complement of SEQ ID NO;

4, SEQ ID NO;

7 and SEQ ID NO;

8, the complement of SEQ ID NO;

7 and the complement of SEQ ID NO;

8, SEQ ID NO;

11 and SEQ ID NO;

12, and the complement of SEQ ID NO;

11 and the complement of SEQ ID NO;

12.

9. A kit comprising:

a pair of polynucleotide primers and a pair of labeled polynucleotide probes selected from the group consisting of;

(1) the pair of primers are two polynucleotides comprising at least 12 consecutive nucleotides of SEQ ID NO;

1 and SEQ ID NO;

2, respectively, and the pair of labeled probes are the pair of labeled nucleic acids of claim 5 wherein the nucleotide sequence is SEQ ID NO;

13;

(2) the pair of primers are two polynucleotides comprising at least 12 consecutive nucleotides of SEQ ID NO;

5 and SEQ ID NO;

6, respectively, and the pair of labeled probes are the pair of labeled nucleic acids of claim 5 wherein the nucleotide sequence is SEQ ID NO;

14; and

(3) the pair of primers are two polynucleotides comprising at least 12 consecutive nucleotides of SEQ ID NO;

9 and SEQ ID NO;

10, respectively, and the pair of labeled probes are the pair of labeled nucleic acids of claim 5 wherein the nucleotide sequence is SEQ ID NO;

15.

10. The kit of claim 9, wherein at least 12 consecutive nucleotides of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

9 and SEQ ID NO;

10 are at least 15 consecutive nucleotides of these sequences.

11. The kit of claim 9, wherein at least 12 consecutive nucleotides of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

9 and SEQ ID NO;

10 are the full length of these sequences.

12. A kit comprising:

an isolated nucleic acid comprising at least 12 consecutive nucleotides of SEQ ID NO;

1;

an isolated nucleic acid comprising at least 12 consecutive nucleotides of SEQ ID NO;

2;

an isolated nucleic acid comprising at least 12 consecutive nucleotides of SEQ ID NO;

5;

an isolated nucleic acid comprising at least 12 consecutive nucleotides of SEQ ID NO;

6;

an isolated nucleic acid comprising at least 12 consecutive nucleotides of SEQ ID NO;

9;

an isolated nucleic acid comprising at least 12 consecutive nucleotides of SEQ ID NO;

10;

a pair of labeled nucleic acids of claim 5 wherein the nucleotide sequence is SEQ ID NO;

13;

a pair of labeled nucleic acids of claim 5 wherein the nucleotide sequence is SEQ ID NO;

14; and

a pair of labeled nucleic acids of claim 5 wherein the nucleotide sequence is SEQ ID NO;

15.

16. A method for detecting a Salmonella species, E. coli O157:

H7, or Listeria monocytogenes comprising the steps of amplifying a nucleotide sequence of a corresponding species using a pair of polynucleotides of claim 9 as PCR primers, and detecting an amplification product.

17. The method of claim 16, wherein the nucleotide sequence is amplified by real-time PCR and the amplification product is detected by fluorescence resonance energy transfer using a pair of labeled polynucleotides of claim 5.

18. The method of claim 17, wherein the nucleotide sequence is amplified by real-time PCR using a pair of polynucleotides of claim 11.

19. The method of claim 17, wherein the amplification product is detected by fluorescence resonance energy transfer using a pair of labeled polynucleotides of claim 8.

20. The method of claim 17, wherein the labeled polynucleotides are included and anneal to the amplification product in the amplification step.

21. The method of claim 17, wherein the detection sensitivity is 50 bacteria or fewer.

13. An isolated nucleic acid comprising a nucleotide sequence selected from SEQ ID NO:

13, SEQ ID NO;

14 and SEQ ID NO;

15.

14. A method for detecting a Salmonella species, E. coli O157:

H7, or Listeria monocytogenes comprising the steps of amplifying a genomic nucleotide sequence comprising nucleotide 9 to nucleotide 243 of SEQ ID NO;

13, nucleotide 7 to nucleotide 354 of SEQ ID NO;

14, or nucleotide 9 to nucleotide 210 of SEQ ID NO;

15, and detecting an amplification product that contains the genomic nucleotide sequence.

15. The method of claim 14, wherein the nucleotide sequence comprises SEQ ID NO:

13, SEQ ID NO;

14 or SEQ ID NO;

15.