Detection of bordetella

• US 7,691,571 B2
• Filed: 01/09/2004
• Issued: 04/06/2010
• Est. Priority Date: 01/31/2001
• 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: Salat, Ulrike, Lohmann, Sabine, Sloan, Lynne M., Patel, Robin, Cockerill, Franklin R. III
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US10/754,223
• Publication Number:
  US 20040265853A1
• Time in Patent Office: 2,279 Days
• Field of Search: None
• 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
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The invention provides methods to detect Bordetella pertussis and/or Bordetella parapertussis in a biological sample. Primers and probes for the differential detection of B. pertussis and B. parapertussis are provided by the invention. Articles of manufacture containing such primers and probes for detecting B. pertussis and/or B. parapertussis are further provided by the invention.

Claims

1. A method for detecting the presence or absence of Bordetella pertussis 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 IS481 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample, wherein said pair of IS481 primers comprises a first IS481 primer and a second IS481 primer, wherein said first IS481 primer consists of the sequence 5′

-CCA GTT CCT CAA GGA CGC-3′

(SEQ ID NO;

1) and said second IS481 primer consists of the sequence 5′

-GAG TTC TGG TAG GTG TGA GCG TA-3′

(SEQ ID NO;

2), wherein said hybridizing step comprises contacting said sample with a pair of IS481 probes, wherein said pair of IS481 probes comprises a first IS481 probe and a second IS481 probe, wherein said first IS481 probe consists of the sequence 5′

-CAC CGC TTT ACC CGA CCT TAC CGC CCA C-3′

(SEQ ID NO;

3) and said second IS481 probe consists of the sequence 5′

-GAC CAA TGG CAA GGC TCG AAC GCT TCA TC-3′

(SEQ ID NO;

11), wherein the members of said pair of IS481 probes hybridize within no more than five nucleotides of each other, wherein a first IS481 probe of said pair of IS481 probes is labeled with a donor fluorescent moiety and a second IS481 probe of said pair of IS481 probes 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 IS481 probe and said corresponding acceptor fluorescent moiety of said second IS481 probe,

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

2. The method of claim 1, wherein the members of said pair of IS481 probes hybridize within no more than two nucleotides of each other.

3. The method of claim 1, wherein the members of said pair of IS481 probes hybridize within no more than one nucleotide of each other.

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

5. The method of claim 1, wherein said acceptor fluorescent moiety is selected from the group consisting of LC™

-Red 640, LC™

-Red 705, Cy5, and Cy5.5.

6. 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 intensity of light emitted by said acceptor fluorescent moiety at said wavelength.

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

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

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

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

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

12. The method of claim 1, wherein the presence of said FRET within 25 cycling steps is indicative of the presence of a B. pertussis infection in said individual.

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

14. The method of claim 13, wherein said preventing comprises performing said amplifying step in the presence of uracil.

15. The method of claim 14, wherein said preventing further comprises treating said biological sample with uracil-DNA glycosylase prior to a first amplifying step.

16. The method of claim 1, wherein said biological sample is selected from the group consisting of nasopharyngeal swabs, nasopharyngeal aspirates, and throat swabs.

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

18. The method of claim 17, wherein said control sample comprises a portion of said IS481 nucleic acid molecule.

19. 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 IS481 primers and IS481 probes, respectively, 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.

20. A method for detecting the presence or absence of Bordetella pertussis and/or B. parapertussis 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 IS481 primers and a pair of IS1001 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample and an IS1001 amplification product if a B. parapertussis IS1001 nucleic acid molecule is present in said sample, wherein said pair of IS481 primers comprises a first IS481 primer and a second IS481 primer, wherein said first IS481 primer consists of the sequence 5′

-CCA GTT CCT CAA GGA CGC-3′

(SEQ ID NO;

     1) and said second IS481 primer consists of the sequence 5′

-GAG TTC TGG TAG GTG TGA GCG TA-3′

(SEQ ID NO;

2), wherein said pair of IS1001 primers comprises a first IS1001 primer and a second IS1001 primer, wherein said first IS1001 primer consists of the sequence 5′

-GGC GAT ATC AAC GGG TGA-3′

(SEQ ID NO;

5) and said second IS1001 primer consists of the sequence 5′

-CAG GGC AAA CTC GTC CAT C-3′

(SEQ ID NO;

6), wherein said hybridizing step comprises contacting said sample with a pair of IS481 probes and a pair of IS1001 probes, wherein said pair of IS481 probes comprises a first IS481 probe and a second IS481 probe, wherein said first IS481 probe consists of the sequence 5′

-CAC CGC TTT ACC CGA CCT TAC CGC CCA C-3′

(SEQ ID NO;

3) and said second IS481 probe consists of the sequence 5′

-GAC CAA TGG CAA GGC TCG AAC GCT TCA TC-3′

(SEQ ID NO;

11), wherein said pair of IS1001 probes comprises a first IS1001 probe and a second IS1001 probe, wherein said first IS1001 probe consists of the sequence 5′

-GCT TGG CAT ACC GTC AAG A-3′

(SEQ ID NO;

12) and said second IS1001 probe consists of the sequence 5′

-GCT GGA CAA GGC TCG-3′

(SEQ ID NO;

13), wherein the members of said pair of IS481 probes hybridize within no more than five nucleotides of each other and wherein the members of said pair of IS1001 probes hybridize within no more than five nucleotides of each other, wherein a first IS481 probe of said pair of IS481 probes is labeled with a donor fluorescent moiety and wherein a second IS481 probe of said pair of IS481 probes is labeled with a corresponding acceptor fluorescent moiety, wherein a first IS1001 probe of said pair of IS1001 probes is labeled with a donor fluorescent moiety and wherein a second IS1001 probe of said pair of IS1001 probes is labeled with a corresponding acceptor fluorescent moiety; and

detecting the presence or absence of FRET between said donor fluorescent moiety of said first IS481 probe and said corresponding acceptor fluorescent moiety of said second IS481 probe and/or between donor fluorescent moiety of said first IS1001 probe and said corresponding acceptor fluorescent moiety of said second IS1001 probe,

wherein the presence of FRET is indicative of the presence of B. pertussis and/or B. parapertussis in said biological sample, and wherein the absence of FRET is indicative of the absence of B. pertussis or B. parapertussis in said biological sample.

21. The method of claim 20, further comprising:

determining the melting temperature between one or both of said IS481 probes and said IS481 amplification product and between one or both of said IS1001 probes and said IS1001 amplification product, wherein said melting temperature(s) confirms said presence or absence of B. pertussis in said sample and said presence or absence of B. parapertussis in said sample.

22. The method of claim 21, wherein said melting temperature(s) distinguish between B. pertussis and B. parapertussis in said sample.

23. The method of claim 20, wherein said acceptor fluorescent moiety of said second IS481 probe and said acceptor fluorescent moiety of said second IS1001 probe are different.

24. A method for detecting the presence or absence of B. pertussis 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 IS481 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample, wherein said pair of IS481 primers comprises a first IS481 primer and a second IS481 primer, wherein said first IS481 primer consists of the sequence 5′

-CCA GTT CCT CAA GGA CGC-3′

(SEQ ID NO;

1) and said second IS481 primer consists of the sequence 5′

-GAG TTC TGG TAG GTG TGA GCG TA-3′

(SEQ ID NO;

2), wherein said hybridizing step comprises contacting said sample with an IS481 probe, wherein the IS481 probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, wherein said IS481 probe is selected from the group consisting of a first IS481 probe consisting of the sequence 5′

-CAC CGC TTT ACC CGA CCT TAC CGC CCA C-3′

(SEQ ID NO;

3) and a second IS481 probe consisting of the sequence 5′

-GAC CAA TGG CAA GGC TCG AAC GCT TCA TC-3′

(SEQ ID NO;

11); and

detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety and said acceptor fluorescent moiety of said IS481 probe,

wherein the presence or absence of FRET is indicative of the presence or absence of B. pertussis in said sample.

25. The method of claim 24, wherein said amplification employs a polymerase enzyme having 5′

to 3′

exonuclease activity.

26. The method of claim 25, wherein said donor and acceptor fluorescent moieties are within no more than 5 nucleotides of each other on said probe.

27. The method of claim 26, wherein said acceptor fluorescent moiety is a quencher.

28. The method of claim 24, wherein said IS481 probe comprises a nucleic acid sequence that permits secondary structure formation, wherein said secondary structure formation results in spatial proximity between said donor and said acceptor fluorescent moiety.

29. The method of claim 28, wherein said acceptor fluorescent moiety is a quencher.