Detection of variola virus

• US 20040029105A1
• Filed: 10/10/2002
• Published: 02/12/2004
• Est. Priority Date: 11/02/2001
• Status: Abandoned Application

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Inventors: Smith, Thomas F., Espy, Mark J.
• Application Number:
  US10/269,018
• Publication Number:
  US 20040029105A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation

Abstract

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

Claims

1. A method for detecting the presence or absence of variola virus 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 hemagglutinin (HA) primers to produce an HA amplification product if a variola virus HA nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of HA probes, wherein the members of said pair of HA probes hybridize within no more than five nucleotides of each other, wherein a first HA probe of said pair of HA probes is labeled with a donor fluorescent moiety and said second HA probe of said pair of HA 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 HA probe and said acceptor fluorescent moiety of said second HA probe,

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

2. The method of claim 1, wherein said pair of HA primers comprises a first HA primer and a second HA primer, wherein said first HA primer comprises the sequence

5′

-CTA ATA TCA TTA GTA TAC GCT ACA C-3′

(SEQ ID NO;

1), and wherein said second HA primer comprises the sequence

5′

-GAG TCG TAA GAT ATT TTA TCC-3′

(SEQ ID NO;

2).

3. The method of claim 1, wherein said first HA probe comprises the sequence

5′

-AAT GAT TAT GTT GTT ATG AGT GCT TG-3′

(SEQ ID NO;

3), and wherein said second HA probe comprises the sequence

5′

-TAT AAG GAG CCC AAT TCC ATT ATT CT-3′

(SEQ ID NO;

4).

4. The method of claim 1, wherein said pair of HA primers comprises a first HA primer and a second HA primer, wherein said first HA primer comprises the sequence

5′

-ATA GTG AAT CGA CTA TAG ACA TAA TA-3′

(SEQ ID NO;

5), and wherein said second HA primer comprises the sequence

5′

-TTG ATT TAG TAG TGA CAA TTC C-3′

(SEQ ID NO;

6).

5. The method of claim 1, wherein said first HA probe comprises the sequence

5′

-CTG TCA CAT ACA CTA GTG ATA TCA TT-3′

(SEQ ID NO;

7), and wherein said second HA probe comprises the sequence

5′

-ATA CAG TAA GTA CAT CAT CTG GAG AA-3′

(SEQ ID NO;

8).

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

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

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

9. The method of claim 1, wherein said corresponding acceptor fluorescent moiety is selected from the group consisting of LC-Red 640, LC-Red 705, Cy5, and Cy5.5.

10. 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.

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

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

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

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

15. The method of claim 1, wherein the presence of said FRET within 50 cycling steps is indicative of the presence of a variola virus infection in said individual.

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

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

18. The method of claim 1, further comprising:

preventing amplification of a contaminant nucleic acid.

19. The method of claim 18, wherein said preventing comprises performing said amplifying step in the presence of uracil.

20. The method of claim 19, wherein said preventing further comprises treating said biological sample with uracil-DNA glycosylase prior to a first amplifying step.

21. The method of claim 1, wherein said biological sample is selected from the group consisting of dermal swabs, cerebrospinal fluid, ganglionic tissue, brain tissue, ocular fluid, blood, sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine.

22. The method of claim 1, wherein said biological sample is autoclaved.

23. The method of claim 1, further comprising:

performing at least one cycling step, wherein said cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of thymidine kinase (TK) primers to produce a TK amplification product if a variola virus TK nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of TK probes, wherein the members of said pair of TK probes hybridize within no more than five nucleotides of each other, wherein a first TK probe of said pair of TK probes is labeled with a donor fluorescent moiety and said second TK probe of said pair of TK 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 TK probe and said acceptor fluorescent moiety of said second TK probe.

24. The method of claim 23, wherein said pair of TK primers comprises a first TK primer and a second TK primer, wherein said first TK primer comprises the sequence

5′

-AAG GAC AGT TCT TTC CAG-3′

(SEQ ID NO;

9),

and wherein said second TK primer comprises the sequence

5′

-TGA TAC ATA TCA TTA CCT CCT A-3′

(SEQ ID NO;

10).

25. The method of claim 23, wherein said first TK probe comprises the sequence

5′

-CCG TTT AAT AAT ATC TTG GAT CTT-3′

(SEQ ID NO;

11), and wherein said second TK probe comprises the sequence

5′

-TTC CAT TAT CTG AAA TGG TGG T-3′

(SEQ ID NO;

12).

26. The method of claim 23, wherein said first TK probe comprises the sequence

5′

-GAC ATT TCA ACG TAA ACC GTT TAA-3′

(SEQ ID NO;

13), and wherein said second TK probe comprises the sequence

5′

-AAT ATC TTG GAT CTT ATT CCA TTA TCT G-3′

(SEQ ID NO;

14).

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

28. The method of claim 27, wherein said control sample comprises said portion of said variola virus HA nucleic acid molecule.

29. 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 HA primers and HA probes, wherein said amplifying step produces a control amplification product, wherein said control probes hybridize to said control amplification product.

30. A method for detecting the presence or absence of variola virus 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 thymidine kinase (TK) primers to produce a TK amplification product if a variola virus TK nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of TK probes, wherein the members of said pair of TK probes hybridize within no more than five nucleotides of each other, wherein a first TK probe of said pair of TK probes is labeled with a donor fluorescent moiety and said second TK probe of said pair of TK 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 TK probe and said acceptor fluorescent moiety of said second TK probe,

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

31. An article of manufacture, comprising:

a pair of hemagglutinin (HA) primers;

a pair of HA probes; and

a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

32. The article of manufacture of claim 31, wherein said pair of HA primers comprise a first HA primer and a second HA primer, wherein said first HA primer comprises the sequence

5′

-CTA ATA TCA TTA GTA TAC GCT ACA C-3′

(SEQ ID NO;

1),

and wherein said second HA primer comprises the sequence

5′

-GAG TCG TAA GAT ATT TTA TCC-3′

(SEQ ID NO;

2).

33. The article of manufacture of claim 31, wherein said pair of HA probes comprises a first HA probe and a second HA probe, wherein said first HA probe comprises the sequence

5′

-AAT GAT TAT GTT GTT ATG AGT GCT TG-3′

(SEQ ID NO;

3),

and wherein said second HA probe comprises the sequence

5′

-TAT AAG GAG CCC AAT TCC ATT ATT CT-3′

(SEQ ID NO;

4).

34. The article of manufacture of claim 31, wherein said pair of HA primers comprise a first HA primer and a second HA primer, wherein said first HA primer comprises the sequence

5′

-ATA GTG AAT CGA CTA TAG ACA TAA TA-3′

(SEQ ID NO;

5),

and wherein said second HA primer comprises the sequence

5′

-TTG ATT TAG TAG TGA CAA TTC C-3′

(SEQ ID NO;

6).

35. The article of manufacture of claim 31, wherein said pair of HA probes comprises a first HA probe and a second HA probe, wherein said first HA probe comprises the sequence

5′

-CTG TCA CAT ACA CTA GTG ATA TCA TT-3′

(SEQ ID NO;

7),

and wherein said second HA probe comprises the sequence

5′

-ATA CAG TAA GTA CAT CAT CTG GAG AA-3′

(SEQ ID NO;

8).

36. The article of manufacture of claim 31, wherein said first HA probe is labeled with said donor fluorescent moiety and wherein said second HA probe is labeled with said corresponding acceptor fluorescent moiety.

37. The article of manufacture of claim 31, further comprising a package insert having instructions thereon for using said pair of HA primers and said pair of HA probes to detect the presence or absence of variola virus in a biological sample.

38. An article of manufacture, comprising:

a pair of thymidine kinase (TK) primers;

a pair of TK probes; and

a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

39. The article of manufacture of claim 38, wherein said pair of TK primers comprises a first TK primer and a second TK primer, wherein said first TK primer comprises the sequence

5′

-AAG GAC AGT TCT TTC CAG-3′

(SEQ ID NO;

9),

and wherein said second TK primer comprises the sequence

5′

-TGA TAC ATA TCA TTA CCT CCT A-3′

(SEQ ID NO;

10).

40. The article of manufacture of claim 38, wherein said pair of TK probes comprises a first TK probe and a second TK probe, wherein said first TK probe comprises the sequence

5′

-CCG TTT AAT AAT ATC TTG GAT CTT-3′

(SEQ ID NO;

11),

and wherein said second TK probe comprises the sequence

5′

-TTC CAT TAT CTG AAA TGG TGG T-3′

(SEQ ID NO;

12).

41. The article of manufacture of claim 38, wherein said pair of TK probes comprises a first TK probe and a second TK probe, wherein said first TK probe comprises the sequence

5′

-GAC ATT TCA ACG TAA ACC GTT TAA-3′

(SEQ ID NO;

13),

and wherein said second TK probe comprises the sequence

5′

-AAT ATC TTG GAT CTT ATT CCA TTA TCT G-3′

(SEQ ID NO;

14).

42. The article of manufacture of claim 38, wherein said first TK probe is labeled with a donor fluorescent moiety and wherein said second TK probe is labeled with an acceptor fluorescent moiety.

43. The article of manufacture of claim 38, further comprising a package insert having instructions thereon for using said pair of TK primers and said pair of TK probes to detect the presence or absence of variola virus in a biological sample.

44. A method for detecting the presence or absence of variola virus 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 hemagglutinin (HA) primers to produce an HA amplification product if a variola virus HA nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with an HA probe, wherein the HA 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 HA probe,

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

45. The method of claim 44, wherein said amplification employs a polymerase enzyme having 5′

to 3′

exonuclease activity.

46. The method of claim 45, wherein said first and second fluorescent moieties are within no more than 5 nucleotides of each other on said probe.

47. The method of claim 45, wherein said second fluorescent moiety is a quencher.

48. The method of claim 44, wherein said HA probe comprises a nucleic acid sequence that permits secondary structure formation, wherein said secondary structure formation results in spatial proximity between said first and second fluorescent moiety.

49. The method of claim 48, wherein said second fluorescent moiety is a quencher.

50. A method for detecting the presence or absence of variola virus 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 dye-binding step, wherein said amplifying step comprises contacting said sample with a pair of hemagglutinin (HA) primers to produce an HA amplification product if a variola virus HA nucleic acid molecule is present in said sample, wherein said dye-binding step comprises contacting said HA amplification product with a double-stranded DNA binding dye; and

detecting the presence or absence of binding of said double-stranded DNA binding dye,

wherein the presence of binding is indicative of the presence of variola virus in said sample, and wherein the absence of binding is indicative of the absence of variola virus in said sample.

51. The method of claim 50, wherein said double-stranded DNA binding dye is selected from the group consisting of SYBRGreenI®

, SYBRGold®

, and ethidium bromide.

52. The method of claim 50, further comprising determining the melting temperature between said HA amplification product and said double-stranded DNA binding dye, wherein said melting temperature confirms said presence or absence of said variola virus.