Detection of group B Streptococcus

• US 8,097,413 B2
• Filed: 07/26/2010
• Issued: 01/17/2012
• 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), Roche Molecular Systems (Roche Holding AG)
• Inventors: Aichinger, Christian, Reiser, Astrid, Uhl, James R, Cockerill, Franklin R III
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US12/843,380
• Publication Number:
  US 20100291582A1
• Time in Patent Office: 540 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 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 a pts amplification product if a GBS pts nucleic acid molecule is present in said sample, wherein said pair of pts primers comprise 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 pts probe, wherein said pts probe is selected from a sequence consisting of 5′

-CAA ATT AAA GAG ACT ATT CGT GCA A-3′

(SEQ ID NO;

3), a sequence consisting of 5′

-CAA GTA AAT GCA GAA ACA GG-3′

(SEQ ID NO;

4), and a sequence that permits secondary structure formation, wherein the pts probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; and

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

wherein the presence or absence of fluorescence is indicative of the presence or absence of GBS in said sample.

2. The method of claim 1, wherein said amplification employs a polymerase enzyme having 5′

to 3′

exonuclease activity.

3. The method of claim 2, wherein said donor and corresponding acceptor fluorescent moieties are within no more than 5 nucleotides of each other on said probe.

4. The method of claim 3, wherein said corresponding acceptor fluorescent moiety is a quencher.

5. The method of claim 1, wherein said corresponding acceptor fluorescent moiety is a quencher.

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

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

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

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

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

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

12. The method of claim 11, wherein said preventing comprises performing said amplifying step in the presence of uracil.

13. The method of claim 12, wherein said preventing further comprises treating said biological sample with uracil-DNA glycosylase prior to a first amplification step.

14. The method of claim 1, wherein said biological sample is selected from the group consisting of anal and/or vaginal swabs.