Multiplex real-time PCR

• US 20030087397A1
• Filed: 07/30/2001
• Published: 05/08/2003
• Est. Priority Date: 01/29/1999
• Status: Abandoned Application

• Current Assignee: Bavarian Nordic Research Institute AS
• Original Assignee: Bavarian Nordic A/S
• Inventors: Klein, Dieter, Salmons, Brian, Gunzburg, Walter H.
• Application Number:
  US09/919,501
• Publication Number:
  US 20030087397A1
• Time in Patent Office: Days
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/702 for retroviruses
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

The present invention relates to a real-time Polymerase Chain Reaction (PCR) method for the detection and quantification of variants of nucleic acid sequences, which differ in the probe-binding site. The method is based on the complete and/or partial amplification of the same region of the variants and the addition of two or more oligonucleotide probes to the same PCR mixture, each probe being specific for the prob-binding site of at least one variant. The method can be applied e.g. to estimate the viral load in a sample, to differentiate between subgroups, subtypes isolates or clades of a viral species or to estimate the impact of the viral load on tumorgenesis.

Claims

1. A real-time Polymerase Chain Reaction (PCR) method for the detection and/or quantification of variants of a nucleic acid sequence, wherein the same region of said variants is completely and/or partially to be amplified, each variant differing in one or more nucleotides within a probe-binding site, said method comprising adding two or more oligonucleotides probes to the same PCR mixture, each probe being specific for the probe binding site of at least one variant.

2. The real-time PCR method according to claim 1, wherein said variants of the nucleic acid sequence differ in one or more nucleotides within the primer binding sites and wherein more than one primer pair is added to the reaction mixture each primer specifically annealing to the primer binding site of at least one subtype.

3. The real-time PCR method according to claim 1, wherein two or more parts of the region are amplified, each part of the region comprising only one probe binding site.

4. The real-time PCR method according to claim 1, wherein the probes are labeled with different fluorescent reporter dyes.

5. The real-time PCR method according to claim 4, wherein the probes are labeled with FAM™

or VIC™

.

6. The real-time PCR method according to claim 1 wherein the nucleic acid sequence is a viral nucleic acid sequence.

7. The real-time PCR method according to claim 6 wherein the viral nucleic acid sequence is a retroviral nucleic acid sequence.

8. The real-time PCR method according to claim 7 wherein the retroviral nucleic acid sequence is a lentiviral nucleic acid sequence.

9. The real-time PCR method according to claim 8 wherein the lentiviral nucleic acid sequence is a Feline Immunodeficiency Viral (FIV) nucleic acid sequence.

10. The real-time PCR method according to claim 9, wherein the probes comprise SEQ ID NO.:

3 and SEQ ID NO.;

6 or SEQ ID NO.;

24.

11. The real-time PCR method according to claim 9, wherein the probes comprise SEQ ID NO.:

3 and SEQ ID NO.;

9 or SEQ ID NO.;

24.

12. The real-time PCR method according to claim 9, wherein a forward primer and a reverse primer are added to the mixture, and the forward primer is selected from the group consisting of;

SEQ ID NO.;

1, SEQ ID NO.;

12, SEQ ID NO.;

22 and combinations thereof, and the reverse primer is selected from the group consisting of;

SEQ ID NO.;

2, SEQ ID NO.;

23 and combinations thereof.

13. The real-time PCR method according to claim 9, wherein a forward primer and a reverse primer are added to the mixture, and the forward primer is selected from the group consisting of:

SEQ ID NO.;

4, SEQ ID NO.;

14, SEQ ID NO.;

15 and combinations thereof, and the reverse primer is selected from the group consisting of;

SEQ ID NO.;

5, SEQ ID NO.;

13 and combinations thereof.

14. The real-time PCR method according to claim 9, wherein a forward primer and a reverse primer are added to the mixture, and the forward primer is selected from the group consisting of:

SEQ ID NO.;

7, SEQ ID NO.;

20, SEQ ID NO.;

21 and combinations thereof, and the reverse primer is SEQ ID NO.;

8.

15. The real-time PCR method according to claim 9, wherein a forward primer and a reverse primer are added to the mixture, and the forward primer is SEQ ID NO.:

16, and the reverse primer is selected from the group consisting of;

SEQ ID NO.;

17, SEQ ID NO.;

19 and combinations thereof.

16. The real-time PCR method according to claim 9, wherein forward primers and reverse primers are added to the reaction mixture, and the forward primers are selected from the group consisting of:

SEQ ID NO.;

1, SEQ ID NO.;

4, SEQ ID NO.;

12, SEQ ID NO.;

14, SEQ ID NO.;

15, SEQ ID NO.;

22 and combinations thereof;

the reverse primers are selected from the group consisting of;

SEQ ID NO.;

2, SEQ ID NO.;

5, SEQ ID NO.;

13, SEQ ID NO.;

23 and combinations thereof; and

the probes are selected from the group consisting of;

SEQ ID NO.;

3, SEQ ID NO.;

6, SEQ ID NO.;

24 and combinations thereof.

17. The real-time PCR method according to claim 9, wherein forward primers and reverse primers are added to the mixture, and the forward primers are selected from the group consisting of:

SEQ ID NO.;

1, SEQ ID NO.;

7, SEQ ID NO.;

12, SEQ ID NO.;

20, SEQ ID NO.;

21, SEQ ID NO.;

22, and the combinations thereof;

the reverse primers are selected from the group consisting of;

SEQ ID NO.;

2, SEQ ID NO.;

8, SEQ ID NO.;

23 and combinations thereof; and

the probes are selected from the group consisting of;

SEQ ID NO.;

3, SEQ ID NO.;

9, SEQ ID NO.;

24 and combinations thereof.

18. The real-time PCR method according claim 1, wherein said PCR is a reverse-transcription (RT) PCR.

19. The real-time PCR method according to claim 1, wherein said variants of nucleic acid sequences are nucleic acid sequences derived from subtypes, isolates, clades or any other subgroup of a species.

20. The real-time PCR method according to claim 1, wherein in the same one-tube reaction a standard nucleic acid sequence is simultaneously amplified and quantified according to real-time PCR principles.

21. The real-time PCR method according to claim 20, wherein the standard nucleic acid sequence is part of a cellular genome.

22. The real-time PCR method according to claim 20, wherein the standard nucleic acid sequence is added in a known copy number to a sample to be tested.

23. The real-time PCR method according to claim 21, wherein the standard nucleic acid sequence derives from the nucleic acid sequence encoding the EGPF (green fluorescence) gene or the 18S rDNA gene.

24. A real-time Polymerase Chain Reaction (PCR) method for the determination of the overall viral load in a sample comprising variants of a viral nucleic acid sequence comprising adding two or more oligonucleotide probes to a PCR mixture, each probe being specific for a probe binding site of at least one of the variants.

25. The method according to claim 24, wherein the variants are derived from nucleic acid sequences derived from subtypes, isolates, clades or any other subgroup of a viral species.

26. A real-time Polymerase Chain Reacton (PCR) method for the determination of the impact of the viral load on tumorgenesis comprising adding two or more oligonucleotide probes to a PCR mixture, each probe being specific for a probe binding site of at lease one viral variant.

27. A real-time Polymerase Chain Reaction (PCR) method for the determination of nucleic acid extraction efficiency or transfection efficiency comprising adding two or more oligonucleotide probes to a PCR mixture, each probe being specific for a probe binding site of a variant.

28. An oligonucleotide probe selected from the group consisting of:

SEQ ID NO.;

6, SEQ ID NO.;

9, SEQ ID NO.;

18, SEQ ID NO.;

24 and complementary strands thereof.

29. A primer selected from the group consisting of:

SEQ ID NO.;

4, SEQ ID NO.;

5, SEQ ID NO.;

7, SEQ ID NO.;

8, SEQ ID NO.;

10, SEQ ID NO.;

17, SEQ ID NO.;

19, SEQ ID NO.;

20, SEQ ID NO.;

21, SEQ ID NO.;

22, and SEQ ID NO.;

23.

30. A set of primers selected from the group consisting of SEQ ID NO.:

2 and SEQ ID NO.;

12.

31. A set of primers selected from the group consisting of:

SEQ ID NO.;

4, SEQ ID NO.;

5, SEQ ID NO.;

13, SEQ ID NO.;

14, SEQ ID NO.;

15 and combinations thereof.

32. A set of primers selected from the group consisting of:

SEQ ID NO.;

7, SEQ ID NO.;

8, SEQ ID NO.;

20, SEQ ID NO.;

21 and combinations thereof.

33. A set of primers selected from the group consisting of:

SEQ ID NO.;

16, SEQ ID NO.;

17, SEQ ID NO.;

19 and combinations thereof.

34. A set of primers selected from the group consisting of:

SEQ ID NO.;

10, SEQ ID NO.;

22, SEQ ID NO.;

23 and combinations thereof.

35. A set of oligonucleotides comprising a primer set selected from the group consisting of SEQ ID NO.:

2, SEQ ID NO.;

4, SEQ ID NO.;

5, SEQ ID NO.;

7, SEQ ID NO.;

8, 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.;

17,SEQ ID NO.;

19,SEQ ID NO.;

20,

SEQ ID NO.;

21, SEQ ID NO.;

22, SEQ ID NO.;

23 and combinations thereof, and a probe selected from the group consisting of;

SEQ ID NO.;

6, SEQ ID NO.;

9, SEQ ID NO.;

18, SEQ ID NO.;

24 and complementary strands thereof.