PRIMERS AND PROBES FOR DETECTING HUMAN PAPILLOMAVIRUS AND HUMAN BETA GLOBIN SEQUENCES IN TEST SAMPLES

• US 20100081124A1
• Filed: 09/30/2008
• Published: 04/01/2010
• Est. Priority Date: 09/30/2008
• Status: Active Grant

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Erickson, Brian J., Abravaya, Klara, Huang, Shihai X., Tang, Ning, Salituro, John A., Mak, Wai-Bing X.
• Granted Patent: US 9,090,948 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates to primers, probes, primer sets, primer and probe sets, methods and kits for detecting human papillomaviruses, human beta globin sequences and human papillomaviruses and human beta globin sequences in a test sample.

Claims

1. A primer for amplifying human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample, wherein the primer has a sequence selected from the group consisting of:

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5 and complements thereof.

2. A probe for detecting HPV types 16, 18, 31, 35, 39, 45, 51, 52, 58, 59 or 66 in a test sample, wherein the probe has a sequence selected from the group consisting of:

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and complements thereof.

3. A primer for amplifying a human beta globin sequence in a test sample, wherein the primer has a sequence of is SEQ ID NO:

6, SEQ ID NO;

7, a complement of SEQ ID NO;

6, a complement of SEQ ID NO;

7 or any combinations thereof.

4. A probe for detecting a human beta globin sequence in a test sample, wherein the probe has a sequence of SEQ ID NO:

22 or a complement thereof.

5. A primer set for amplifying HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample, wherein said primer set comprises:

(a) at least one forward primer having a sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, complements thereof and any combinations thereof; and

(b) at least one reverse primer having a sequence selected from the group consisting of SEQ ID NO;

4, SEQ ID NO;

5, complements thereof and any combinations thereof.

6. The primer set of claim 5, wherein said primer set comprises forward primers having the sequence of SEQ ID NO:

1, SEQ ID NO;

2 and SEQ ID NO;

3 or complements thereof and reverse primers having the sequence of SEQ ID NO;

4 and SEQ ID NO;

5 or complements thereof.

7. A primer and probe set for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample, wherein the primer and probe set comprises:

(a) three forward primers having a sequence of;

SEQ ID NO;

1, SEQ ID NO;

2 and SEQ ID NO;

3 or complements thereof and two reverse primers having a sequence of;

SEQ ID NO;

4 and SEQ ID NO;

5 or complements thereof; and

(b) fourteen probes having a sequence of;

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21 or complements thereof.

8. A primer and probe set for detecting human beta globin in a test sample, wherein the primer and probe set comprises:

(a) a forward primer having a sequence of SEQ ID NO;

6 or a complement thereof and a reverse primer having a sequence of SEQ ID NO;

7 or a complement thereof; and

(b) a probe having a sequence of SEQ ID NO;

22 or a complement thereof.

13. The method of claim 9, further comprising:

(a) contacting the test sample with the primers from the primer and probe set of claim 8 under amplification conditions to generate a second target sequence; and

(b) detecting hybridization between the second target sequence and the probe from the primer and probe set of claim 8 as an indication of the presence of a human beta globin in the test sample.

15. A method for detecting human beta globin in a test sample, the method comprising the steps of:

(a) contacting the test sample with the primers from the primer and probe set of claim 8 under amplification conditions to generate a first target sequence; and

(b) detecting hybridization between the first target sequence and the probe from the primer and probe set of claim 8 as an indication of the presence of a human beta globin in the test sample.

16. The method of claim 15, wherein the probe is labeled with a detectable label.

17. The method of claim 16, wherein the detectable label comprises a fluorescent moiety attached at the 5′

end of the probe.

18. The method of claim 17, wherein the probe further comprises a quencher moiety attached at its 3′

end.

19. The method of claim 15, further comprising:

(a) contacting the test sample with primers from the primer and probe set of claim 7 under amplification conditions to generate a second target sequence; and

(b) detecting hybridization between the second target sequence and at least one probe from the primer and probe set of claim 7 as an indication of the presence of one or more of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample.

9. A method for detecting one or more of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample, the method comprising the steps of:

(a) contacting the test sample with three forward primers having a sequence of;

SEQ ID NO;

1, SEQ ID NO;

2 and SEQ ID NO;

3 or complements thereof and two reverse primers having a sequence of;

SEQ ID NO;

4 and SEQ ID NO;

5 or complements thereof under amplification conditions to generate a first target sequence; and

(b) detecting hybridization between the first target sequence and at least one probe as an indication of the presence of one or more of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample, wherein the probe has a sequence of;

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21 or a complement thereof.

10. The method of claim 9, wherein at least one probe is labeled with a detectable label.

11. The method of claim 10, wherein the detectable label comprises a fluorescent moiety attached at the 5′

end of at least one probe.

12. The method of claim 11, wherein at least one probe further comprises a quencher moiety attached at its 3′

end.

14. A method for detecting HPV types 16, 18 or 16 and 18 in a test sample, the method comprising the steps of:

(a) contacting the test sample with three forward primers having a sequence of;

SEQ ID NO;

1, SEQ ID NO;

2 and SEQ ID NO;

3 or complements thereof and two reverse primers having a sequence of;

SEQ ID NO;

4 and SEQ ID NO;

5 or complements thereof under amplification conditions to generate a first target sequence; and

(b) detecting hybridization between the first target sequence and the following;

(i) a first probe having a sequence of SEQ ID NO;

8 or a complement thereof as an indication of the presence of HPV type 16, wherein said first probe is labeled with a first detectable label;

(ii) a second probe having a sequence of SEQ ID NO;

9 or a complement thereof as an indication of the presence of HPV type 18, wherein said second probe is labeled with a second detectable label, and further wherein the second detectable label is a different detectable label than the first detectable label;

(iii) one or more additional probes having a sequence of;

SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21 or a complement thereof as an indication of the presence of HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 or 68, wherein each of the one or more additional probes is labeled with an identical third detectable label and further wherein said third detectable label is a different detectable label than the first detectable label and the second detectable label.

20. A kit for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample, wherein said kit comprises:

(a) at least one forward primer having a sequence selected from the group consisting of;

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, complements thereof and any combinations thereof;

(b) at least one reverse primer having a sequence selected from the group consisting of;

SEQ ID NO;

4, SEQ ID NO;

5, complements thereof and any combinations thereof; and

(c) amplification reagents.

21. The kit of claim 20, wherein the kit further comprises at least one probe, wherein the probe is selected from the group consisting of:

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21 and complements thereof.

22. The kit of claim 20, wherein the kit further comprises probes having the sequence of:

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21 or complements thereof.

23. A primer and probe kit for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample, wherein said kit comprises:

(a) three forward primers having a sequence of;

SEQ ID NO;

1, SEQ ID NO;

2 and SEQ ID NO;

3 or complements thereof and two reverse primers having a sequence of;

SEQ ID NO;

4 and SEQ ID NO;

5 or complements thereof;

(b) fourteen probes having a sequence of;

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21 or complements thereof; and

(c) amplification reagents.

24. The primer and probe kit of claim 23, wherein the kit further comprises a forward primer having a sequence of SEQ ID NO:

6 or a complement thereof and a reverse primer having a sequence of SEQ ID NO;

7 or a complement thereof and a probe having a sequence of SEQ ID NO;

22 or a complement thereof for detecting human beta globin in the test sample.

25. A primer and probe kit for detecting human beta globin in a test sample, wherein said kit comprises:

(a) a forward primer having a sequence of SEQ ID NO;

6 or complement thereof and a reverse primer having a sequence of SEQ ID NO;

7 or a complement thereof;

(b) a probe having a sequence of SEQ ID NO;

22 or a complement thereof; and

(c) amplification reagents.

26. The primer and probe kit of claim 25, wherein the kit further comprises:

(d) three forward primers having a sequence of;

SEQ ID NO;

1, SEQ ID NO;

2 and SEQ ID NO;

3 or complements thereof and two reverse primers having a sequence of;

SEQ ID NO;

4 and SEQ ID NO;

5 or complements thereof; and

(e) fourteen probes having a sequence of;

SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

1, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20 and SEQ ID NO;

21 or complements thereof, for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample.