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

• US 7,241,566 B2
• Filed: 06/21/2002
• Issued: 07/10/2007
• Est. Priority Date: 06/22/2001
• Status: Expired due to Fees

• Current Assignee: Marshfield Clinic
• Original Assignee: Marshfield Clinic
• Inventors: Vevea, Dirk N., Ellingson, Jay L. E.
• Primary Examiner(s): Myers; Carla J.
• Assistant Examiner(s): SALMON, KATHERINE D
• Application Number:
  US10/178,331
• Publication Number:
  US 20030022214A1
• Time in Patent Office: 1,845 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.11
• 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 selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, and the complements thereof.

2. A kit for detecting the presence of E. coli O157:

H7, said kit comprising a primer pair and a pair of labeled probes, wherein the primer pair consists of SEQ ID;

5 and SEQ ID NO;

6 or the complements thereof, and the pair of labeled probes consists of a first probe consisting of SEQ ID NO;

7, or the complement thereof, and a label at the 3′

end and a second probe consisting of SEQ ID NO;

8, or the complement thereof, and a label at the 5′

end.

3. The kit of claim 2, wherein the first probe is labeled with fluorescein and the second probe is labeled with LC RED 640 or LC RED 705.

4. A method for detecting the presence of E. coli O157:

H7, said method comprising;

(a) obtaining a sample and amplifying at least a portion of the eae gene of E. coli O157;

H7 with a primer pair consisting of SEQ ID NO;

5 and SEQ ID NO;

6 or the complements thereof in the presence of a pair of labeled probes such that said amplification step produces an amplified eae gene product if the sample comprises at least a portion of the eae gene of E. Coli O 157;

H7 that comprises the nucleotide sequence of the primer pair, wherein the pair of labeled probes consists of a first probe consisting of SEQ ID NO;

7, or the complement thereof, and a donor fluorophore of a fluorescence resonance energy transfer pair at the 3′

end and a second probe consisting of SEQ ID NO;

8, or the complement thereof, and an acceptor fluorophore of a fluorescence resonance energy transfer pair at the 5′

end, such that upon hybridization of the first and second probes with the amplified eae gene product, the donor and the acceptor fluorophores are less than 6 nucleotides apart from one another; and

(b) detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorophore and the acceptor fluorophore,

wherein the presence of FRET is indicative of the presence of E. coli O157;

H7 in the sample.

5. The method of claim 4, wherein the donor fluorophore is fluoroscein.

6. The method of claim 4, wherein the acceptor fluorophore is LC RED 640 or LC RED 705.