Multiplexed methods for detecting target rnas
AssigneePresident and Fellows of Harvard College
InventorsMarc W. KIRSCHNER, Mingjie DAI, George M. CHURCH
Life of this patent
Estimating the term…
Abstract
The technology described herein is directed to methods, kits, compositions, and systems for detecting a target RNA, such as a small amount of viral RNA. In one aspect, described herein are methods of detecting the target RNA, using primers comprising at least one barcode region. In other aspects, described herein are kits, compositions, and systems suitable to practice the methods described herein to detect the target RNA.
Claims36
A multiplexed method of detecting at least one target RNA in at least two samples, comprising:
- a) contacting the at least two samples with a reverse transcriptase and a first primer or first set of primers comprising at least a first barcode, under conditions permitting the generation of reverse transcription products;
- b) combining reverse transcription products from samples in step (a) in one container to form a pooled reverse transcription product mixture;
- c) contacting the pooled reverse transcription product mixture with a DNA polymerase and a second set of primers under conditions permitting the generation of amplification products;
- and
- d) sequencing the amplification products, thereby detecting at least one target RNA, if present, in the at least two samples.
(canceled)
5-6. (canceled)
8-9. (canceled)
11-13. (canceled)
(canceled)
18-22. (canceled)
24-25. (canceled)
27-58. (canceled)
(canceled)
62-64. (canceled)
67-81. (canceled)
84-98. (canceled)
100-108. (canceled)
110-113. (canceled)
A reverse transcription solution comprising:
- a) a reverse transcriptase;
- b) a first set of primers comprising at least one barcode;
- c) a detergent;
- d) carrier nucleic acid;
- e) at least one positive control nucleic acid;
- f) at least one stabilization agent;
- and/or
- g) reverse transcription reaction buffer.
(canceled)
A kit for detecting a target RNA in a sample, comprising:
- a) a reverse transcriptase;
- b) a first set of primers comprising at least one barcode;
- c) a detergent;
- d) a carrier nucleic acid;
- e) a positive control nucleic acid;
- f) at least one stabilization agent;
- g) at least two containers;
- h) a DNA polymerase;
- i) a second set of primers;
- j) Uracil-DNA Glycosylase (UDG) enzyme;
- k) a protector nucleic acid;
- and/or
- l) a third set of primers.
117-118. (canceled)
Dependencies are inferred from the claim text.