Non-target amplification method for detection of RNA splice-forms in a sample

• US 9,797,000 B2
• Filed: 04/30/2010
• Issued: 10/24/2017
• Est. Priority Date: 05/01/2009
• Status: Expired due to Fees

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Lowe, Brian, Fulbright, Anna K., Nazarenko, Irina
• Primary Examiner(s): Woolwine, Samuel
• Application Number:
  US12/771,042
• Publication Number:
  US 20100311039A1
• Time in Patent Office: 2,734 Days
• Field of Search: None
• 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 non-target amplification reverse hybrid capture method of detecting the presence of a target human papillomavirus (HPV) RNA, the method comprising:

(a) providing the target human HPV RNA by a method comprising;

(i) incubating a biological sample comprising a cell containing the target HPV RNA with carboxyl beads under conditions sufficient for the cell to bind to the carboxyl beads;

(ii) isolating the beads;

(iii) lysing the cell attached to the isolated beads; and

(iv) isolating the beads from the lysed biological sample, wherein the resulting supernatant contains the target HPV RNA for detection;

(b) providing at least one DNA capture probe, wherein the at least one DNA capture probe 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) isolating the target RNA;

DNA capture probe complex by removal of non-target RNA sequences;

(e) providing a first combination of DNA amplification probes, and hybridizing said first combination of DNA amplification probes to said target RNA;

DNA capture probe complex, yielding a target RNA;

DNA capture/amplification probe complex, wherein said DNA amplification probes are from about 15 to about 200 bases in length and are designed to be complementary over the length of the target HPV RNA and combined in mixtures to cover specific genes, excluding regions that are already covered by the at least one DNA capture probe, in order to allow detection of specific splice forms of the RNA;

(f) 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;

(g) detecting said antibody, and comparing the detection results with results produced from a different combination of DNA amplification probes, wherein the comparing indicates the presence of a particular RNA splice-form,

wherein the at least one DNA capture probe and the DNA amplification probes are complementary to RNA from HPV, and

wherein said carboxyl beads do not comprise an immobilized ligand.

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 at least one DNA capture probe is from about 15 to about 200 bases in length.

8. The method of claim 1, 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.

9. The method of claim 1, wherein the first combination of DNA amplification probes and the different combination of DNA amplification probes comprise degenerate signal amplification probes that cover predicted splice form sequences.