NON-TARGET AMPLIFICATION METHOD FOR DETECTION OF RNA SPLICE-FORMS IN A SAMPLE

• US 20100311039A1
• Filed: 04/30/2010
• Published: 12/09/2010
• Est. Priority Date: 05/01/2009
• Status: Active Grant

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Lowe, Brian, Fulbright, Anna K., Nazarenko, Irina
• Granted Patent: US 9,797,000 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• 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 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides

Abstract

Provided are methods of isolating RNA from a biological sample, methods and means for determining the presence of particular RNA splice-form variants in a biological sample, methods and means for determining the relative ratio of RNA ratios in a biological sample, and methods and means for predicting the progression of precancerous cervical lesions.

Claims

1. A method of detecting the presence of a target RNA, the method comprising:

a) providing at least one DNA capture probe, wherein the at least one DNA capture probe is bound to a support;

b) hybridizing the target RNA to said at least one DNA capture probe, yielding a target RNA;

DNA capture probe complex;

c) isolating the target RNA;

DNA capture probe complex;

d) providing at least one DNA amplification probe, and hybridizing said at least one DNA amplification probe to said target RNA;

DNA capture probe complex, yielding a target RNA;

DNA capture/amplification probe complex;

e) providing an anti-RNA;

DNA hybrid antibody, and incubating said target RNA;

DNA capture/amplification probe complex with said antibody, yielding a target RNA;

DNA;

antibody complex;

f) detecting said antibody, wherein said detecting indicates the presence of said target RNA.

2. The method of claim 1, wherein said antibody is conjugated to a detectable marker, and wherein said detecting comprises detecting the marker.

3. The method of claim 2, wherein the detectable marker is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

4. The method of claim 1, wherein said detecting comprises providing a second antibody that binds to said anti-RNA:

DNA hybrid antibody, wherein said second antibody is conjugated to a detectable marker, and wherein said detecting further comprises detecting the marker.

5. The method of claim 1 wherein the support comprises a magnetic bead.

6. The method of claim 5, wherein said at least one DNA capture probe is conjugated to a biotin molecule, and wherein said support is conjugated to at least one streptavidin molecule.

7. The method of claim 1, wherein the target RNA is from virus, bacteria, mycobacteria or plasmodia.

8. The method of claim 1, wherein the target RNA is from Herpesviridae, human immunodeficiency virus, Chlamydia spp., Neisseria spp., Staphylococcus aureus, mycobacteria, SARS coronavirus, Orthomixoviridae, or Papillomaviridae.

9. The method of claim 1, wherein the at least one DNA capture probe and the at least one DNA amplification probe are from about 15 to about 200 bases in length.

10. The method of claim 1, wherein the target RNA is a splice variant, and wherein the at least one DNA capture probe and the at least one DNA amplification probe are selected to detect the presence of said splice variant.

11. The method of claim 11, wherein the at least one DNA capture probe and the at least one DNA amplification probe are complementary to RNA from HPV high risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 66, 73, and 82.

12. A kit for the detection of a target RNA, the kit comprising:

a) at least One DNA capture probe, bound to a magnetic support;

b) at least one DNA amplification probe;

c) an anti-RNA;

DNA hybrid antibody; and

d) a detection reagent.

13. The kit of claim 12, wherein said anti-RNA:

DNA hybrid antibody is conjugated to a detectable marker, and wherein said detection reagent comprises a substrate for said detectable marker.

14. The kit of claim 12, further comprising a second antibody that binds to said anti-RNA:

DNA hybrid antibody, wherein said second antibody is conjugated to a detectable marker, and wherein said detection reagent comprises a substrate for said detectable marker.

15. A method of providing target RNA for detection, the method comprising:

a) incubating a biological sample containing the target RNA with carboxyl beads;

b) isolating the beads;

c) lysing the biological sample attached to the isolated beads; and

d) isolating the beads from the lysed biological sample, wherein the resulting supernatant contains the target RNA for detection.

16. A method of detecting the presence of a target RNA, the method comprising:

a) providing at least one DNA capture probe;

b) providing a first anti-RNA;

DNA hybrid antibody, wherein the first anti-RNA;

DNA hybrid antibody is bound to a support;

c) hybridizing the target RNA to said at least one DNA capture probe, yielding a target RNA;

DNA capture probe complex;

d) incubating said target RNA;

DNA capture probe complex with said anti-RNA;

DNA hybrid antibody, yielding a bound target RNA;

DNA capture probe complex;

e) providing at least one DNA amplification probe, and hybridizing said at least one DNA amplification probe to said bound target RNA;

DNA capture probe complex, yielding a bound target RNA;

DNA capture/amplification probe complex;

f) providing a second anti-RNA;

DNA hybrid antibody, and incubating said bound target RNA;

DNA capture/amplification probe complex with said second anti-RNA;

DNA hybrid antibody, yielding a bound target RNA;

DNA;

antibody complex;

g) detecting said second anti-RNA;

DNA hybrid antibody, wherein said detecting indicates the presence of said target RNA.

17. The method of claim 16, wherein said second anti-RNA:

DNA hybrid antibody is conjugated to a detectable marker, and wherein said detecting comprises detecting the marker.

18. The method of claim 17, wherein the detectable marker is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.

19. The method of claim 16, wherein said detecting comprises providing a third antibody that binds to said second anti-RNA:

DNA hybrid antibody, wherein said third antibody is conjugated to a detectable marker, and wherein said detecting further comprises detecting the marker.

20. The method of claim 16 wherein the support comprises a magnetic bead.

21. The method of claim 20, wherein said first anti-RNA:

DNA hybrid antibody is conjugated to a biotin molecule, and wherein said support is conjugated to at least one streptavidin molecule.

22. The method of claim 16, wherein the target RNA is from virus, bacteria, mycobacteria or plasmodia.

23. The method of claim 16, wherein the target RNA is from Herpesviridae, human immunodeficiency virus, Chlamydia spp., Neisseria spp., Staphylococcus aureus, mycobacteria, SARS coronavirus, Orthomixoviridae, or Papillomaviridae.

24. The method of claim 16, wherein the at least one DNA capture probe and the at least one DNA amplification probe are from about 15 to about 200 bases in length.

25. The method of claim 16, wherein the target RNA is a splice variant, and wherein the at least one DNA capture probe and the at least one DNA amplification probe are selected to detect the presence of said splice variant.

26. The method of claim 25, wherein the at least one DNA capture probe and the at least one DNA amplification probe are complementary to RNA from HPV high risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 66, 73, and 82.