Detection of group B streptococcus

• US 7,427,475 B2
• Filed: 11/18/2003
• Issued: 09/23/2008
• Est. Priority Date: 11/18/2003
• Status: Active Grant

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic), Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Inventors: Reiser, Astrid, Uhl, James R., Cockerill, Franklin R. III, Aichinger, Christian
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US10/716,005
• Publication Number:
  US 20050106578A1
• Time in Patent Office: 1,771 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The invention provides methods to detect group B streptococcus (GBS) in biological samples using real-time PCR. Primers and probes for the detection of GBS are provided by the invention. Articles of manufacture containing such primers and probes for detecting GBS are further provided by the invention.

Claims

1. A method for detecting the presence or absence of Group B Streptococcus (GBS) in a biological sample from an individual, said method comprising:

performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of pts primers to produce an pts amplification product if a GBS pts nucleic acid molecule is present in said sample, wherein said pair of pts primers comprises a first pts primer and a second pts primer, wherein said first pts primer consists of the sequence 5′

-TGA GAA GGC AGT AGA AAG CTT AG-3′

(SEQ ID NO;

1) and wherein said second pts primer consists of the sequence 5′

-TGC ATG TAT GGG TTA TCT TCC-′

3 (SEQ ID NO;

2), wherein said hybridizing step comprises contacting said sample with a pair of pts probes, wherein said pair of pts probes comprises a first pts probe and a second pts probe, wherein said first pts probe consists of the sequence 5′

-CAA ATT AAA GAG ACT ATT CGT GCA A-3′

(SEQ ID NO;

3) and wherein said second pts probe consists of the sequence 5′

-CAA GTA AAT GCA GAA ACA GG-3′

(SEQ ID NO;

4), wherein the members of said pair of pts probes hybridize to said amplification product within no more than five nucleotides of each other, wherein said first pts probe is labeled with a donor fluorescent moiety and wherein said second pts probe is labeled with a corresponding acceptor fluorescent moiety; and

detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first pts probe and said acceptor fluorescent moiety of said second pts probe,

wherein the presence of FRET is indicative of the presence of GBS in said biological sample, and wherein the absence of FRET is indicative of the absence of GBS in said biological sample.

2. The method of claim 1, wherein the presence of said FRET within 45 cycling steps is indicative of the presence of a GBS infection in said individual.

3. The method of claim 1, wherein the presence of said FRET within 40 cycling steps is indicative of the presence of a GBS infection in said individual.

4. The method of claim 1, wherein the presence of said FRET within 30 cycling steps is indicative of the presence of a GBS infection in said individual.

5. The method of claim 1, wherein said cycling step is performed on a control sample.

6. The method of claim 5, wherein said control sample comprises said GBS pts nucleic acid molecule.

7. The method of claim 1, wherein said cycling step uses a pair of control primers and a pair of control probes, wherein said control primers and said control probes are other than said pts primers and said pts probes, wherein a control amplification product is produced if control template is present in said sample, wherein said control probes hybridize to said control amplification product.

8. The method of claim 1, wherein said donor fluorescent moiety is fluorescent.

9. The method of claim 1, wherein said detecting step comprises exciting said biological sample at a wavelength absorbed by said donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by said acceptor fluorescent moiety.

10. The method of claim 1, wherein said detecting comprises quantitating said FRET.

11. The method of claim 1, wherein said detecting step is performed after each cycling step.

12. The method of claim 1, wherein said detecting step is performed in real time.

13. The method of claim 1, further comprising determining the melting temperature between one or both of said probe(s) and said amplification product, wherein said melting temperature confirms said presence or said absence of said GBS.

14. The method of claim 1, further comprising preventing amplification of a contaminant nucleic acid.

15. The method of claim 14, wherein said preventing comprises performing said amplifying step in the presence of uracil.

16. The method of claim 15, wherein said preventing further comprises treating said biological sample with uracil-DNA glycosylase prior to a first amplification step.

17. The method of claim 1, wherein said biological sample is selected from anal swabs, vaginal swabs, or anal and vaginal swabs.