Method for detecting and quantifying latent retroviral RNA species

Patent metadata

Field	Value
Publication number	US-11193176-B2
Application number	US-201414587365-A
Country	US
Kind code	B2
Filing date	Dec 31, 2014
Priority date	Dec 31, 2013
Publication date	Dec 7, 2021
Grant date	Dec 7, 2021

Abstract

The invention includes methods for determining the presence of a latent viral population by analyzing an RNA population from the virus with digital techniques, such as digital PCR or by sequencing cDNA produced from the RNA. The invention additional includes methods for determining the presence of latent viral populations by detecting and/or quantifying enzymes that are uniquely associated with the virus, e.g., reverse transcriptases.

First claim

What is claimed is:

1. A method for quantifying reservoirs of replication-competent latent retrovirus in cells, comprising the steps of:
providing cells containing latent retrovirus;
activating the cells without culturing the cells with any other cells;
purifying virion particles from the activated cells;
isolating one or more of the virion particles in respective aqueous droplets surrounded by an immiscible fluid, one or more of the droplets comprising a reverse transcriptase, a polymerase, a primer capable of hybridizing to retroviral RNA, a barcode sequence, a primer for amplifying retroviral cDNA, and a probe comprising a detectable reporter;
lysing the virion particles to release RNA molecules within the droplets;
reverse transcribing the RNA molecules into cDNAs and incorporating the barcode sequence into the cDNA product during cDNA synthesis;
amplifying the cDNAs with the polymerase to form amplicons comprising substantially identical copies of the cDNAs in the presence of probes that include the detectable reporter;
detecting the amplicons hybridized to the probe comprising the detectable reporter, wherein only a single detectable reporter is used in any droplet; and
quantifying the replication-competent latent retrovirus by digitally counting aqueous droplets containing the detected amplicons; and
sequencing the substantially identical copies of the cDNA product.

2. The method of claim 1 , wherein the detectable reporter is the same fluorescent label on each of the probes and copies of cDNA are detected with fluorescence detection.

3. The method of claim 1 , further comprising introducing into the droplet an amount of exogenous synthetic RNA, DNA, or mRNA.

4. The method of claim 3 , further comprising normalizing the detected cDNA against the exogenous synthetic RNA, DNA, or mRNA.

5. The method of claim 1 , wherein the cells are T-cells and wherein the activating step includes exposing the cells to a mitogen.

6. The method of claim 1 , wherein the latent retrovirus is a human immunodeficiency virus (HIV).

7. A method for quantifying reservoirs of replication-competent latent retrovirus in cells, comprising the steps of:
providing cells containing latent retrovirus;
activating the cells with a mitogen without culturing the cells with irradiated PBMCs;
concentrating or purifying the virion particles;
isolating virion particles within aqueous droplets surrounded by an immiscible fluid, one or more of the droplets comprising a reverse transcriptase, a polymerase, a barcode sequence, a primer to amplify retroviral cDNA, and a retroviral RNA-specific primer;
lysing the virion particles within the droplets to release viral RNA such that individual viral RNAs are droplet encapsulated;
synthesizing with the reverse transcriptase a cDNA product from the viral RNA and incorporating the barcode sequence into the cDNA product during cDNA synthesis;
amplifying the cDNA product with the polymerase to form amplicons;
sequencing the amplicons to identify amplicons having the barcode sequence; and
quantifying the replication-competent latent retrovirus by counting aqueous droplets containing the identified amplicons.

8. The method of claim 7 , wherein the barcode sequence comprises a random barcode.

9. The method of claim 7 , wherein the barcode sequence is unique to the droplet.

10. The method of claim 7 , wherein the barcode sequence comprises from about 4 to about 20 bases and uniquely encodes the virion in the droplet.

11. The method of claim 7 , wherein the barcode sequence has no homopolymer regions.

12. The method of claim 7 , wherein molecular barcodes are incorporated into cDNAs for each of the virion particles within the aqueous droplets.

Assignees

• Bio Rad Laboratories

Inventors

• Samuels Michael
• Link Darren R
• Banerjee Sirshendu Roopom

Classifications

• C12Q1/702 Primary

  for retroviruses · CPC title

• C12Q1/70

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title

• C12Q2563/159

  Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components · CPC title