Methods and oligonucleotides for the detection of Salmonella SP., E coli 0157:H7, and Listeria monocytogenes

• US 20060240442A1
• Filed: 04/20/2005
• Published: 10/26/2006
• Est. Priority Date: 04/20/2005
• Status: Abandoned Application

• Current Assignee: Dirk N. Vevea
• Original Assignee: Dirk N. Vevea
• Inventors: Vevea, Dirk N.
• Application Number:
  US11/110,510
• Publication Number:
  US 20060240442A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A method for detecting a Salmonella species, E. coli 0157: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. A method for detecting a food-borne pathogenic bacteria located within a food sample, the method comprising the steps of:

providing a food sample containing a target DNA sequence;

providing a real-time polymerase chain reaction (PCR) primer containing a pair of nucleotide sequences;

amplifying said target DNA sequence with said PCR primer;

detecting said target DNA sequence utilizing a hybridization probe, said hybridization probe containing at least one nucleotide sequence, said nucleotide sequence located in said hybridization probe being compatible with said PCR primer nucleotide sequence;

detecting the presence of said food-borne bacteria located within said food sample, said detection carried out by amplifying said PCR primer and said hybridization probe with a nucleotide sequence compatible with said PCR primer nucleotide sequences and said hybridization probe nucleotide sequence, said detection being obtainable within 12 hours of commencement of the method.

2. The method according to claim 1 wherein said food-borne pathogenic bacteria is Salmonella sp.

3. The method according to claim 2 wherein said nucleotide sequences of said PCR primer are selected from the group consisting of:

SEQ ID NO;

1, SEQ ID NO;

2, and the complements of SEQ ID NO;

1 and SEQ ID NO;

2.

4. The method according to claim 3 wherein said nucleotide sequence of said hybridization probe is selected form the group consisting of:

SEQ ID NO;

3, SEQ ID NO;

4 and the complements of SEQ ID NO;

3 and SEQ ID NO;

5. The method according to claim 4 wherein said amplification nucleotide sequence is selected from the group consisting of:

SEQ ID NO;

13 and the complements of SEQ ID NO;

13.

6. The method according to claim 1 wherein said food-borne pathogenic bacteria is Listeria monocytogenes.

7. The method according to claim 6 wherein said nucleotide sequence of said PCR primer is selected from the group consisting of:

SEQ ID NO;

9, SEQ ID NO;

10, and the complements of SEQ ID NO;

9 and SEQ ID NO;

10.

8. The method according to claim 7 wherein said nucleotide sequence of said hybridization probe is selected form the group consisting of:

SEQ ID NO;

11, SEQ ID NO;

12 and the complements of SEQ ID NO;

11 and SEQ ID NO;

12.

9. The method according to claim 8 wherein said amplification nucleotide sequence is selected from the group consisting of:

SEQ ID NO;

15 and the complements of SEQ ID NO;

15.

10. The method according to claim 1 wherein said step of detecting said amplified target DNA is performed by fluorescence resonance energy transfer (FRET).

11. A method for detecting a food-borne pathogenic bacteria located within a food sample, the method comprising the steps of:

providing a food sample containing a target DNA sequence;

providing a real-time polymerase chain reaction (PCR) primer containing a pair of nucleotide sequences;

amplifying said target DNA sequence with said PCR primer;

detecting said target DNA sequence utilizing a hybridization probe, said hybridization probe containing at least one nucleotide sequence, said nucleotide sequence located in said hybridization probe being compatible with said PCR primer nucleotide sequences;

detecting the presence of said food-borne bacteria located within said food sample, said detection carried out by amplifying said PCR primer and said hybridization probe with a nucleotide sequence compatible with said PCR primer nucleotide sequences and said hybridization probe nucleotide sequence, said detection being performed within 6 hours of commencement of the method.

12. The method according to claim 11 wherein said food-borne pathogenic bacteria is Salmonella sp.

13. The method according to claim 12 wherein said nucleotide sequences of said PCR primer are selected from the group consisting of:

SEQ ID NO;

1, SEQ ID NO;

2, and the complements of SEQ ID NO;

1 and SEQ ID NO;

2.

14. The method according to claim 13 wherein said nucleotide sequence of said hybridization probe is selected form the group consisting of:

SEQ ID NO;

3, SEQ ID NO;

4 and the complements of SEQ ID NO;

3 and SEQ ID NO;

4.

15. The method according to claim 14 wherein said amplification nucleotide sequence is selected from the group consisting of:

SEQ ID NO;

13 and the complements of SEQ ID NO;

13.

16. The method according to claim 11 wherein said food-borne pathogenic bacteria is Listeria monocytogenes.

17. The method according to claim 16 wherein said nucleotide sequence of said PCR primer is selected from the group consisting of:

SEQ ID NO;

9, SEQ ID NO;

10, and the complements of SEQ ID NO;

9 and SEQ ID NO;

10.

18. The method according to claim 17 wherein said nucleotide sequence of said hybridization probe is selected form the group consisting of:

SEQ ID NO;

11, SEQ ID NO;

12 and the complements of SEQ ID NO;

11 and SEQ ID NO;

12.

19. The method according to claim 18 wherein said amplification nucleotide sequence is selected from the group consisting of:

SEQ ID NO;

15 and the complements of SEQ ID NO;

15.

20. The method according to claim 11 wherein said step of detecting said amplified target DNA is performed by fluorescence resonance energy transfer (FRET).