Method for detecting and quantifying latent retroviral rna species

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.

What is claimed is: 
     
         1 . A method for detection of latent retrovirus, comprising the steps of:
 isolating a virion particle in an aqueous droplet surrounded by an immiscible fluid;   lysing the virion particle in the aqueous droplet to release an RNA molecule;   exposing the RNA molecule to at least one primer capable of hybridizing to at least a portion of the RNA molecule;   synthesizing a cDNA product from the RNA molecule; and   detecting the cDNA product.   
     
     
         2 . The method of  claim 1 , wherein detecting comprises amplifying the cDNA product to produce a plurality of substantially identical copies of the cDNA product. 
     
     
         3 . The method of  claim 2 , further comprising sequencing the substantially identical copies of the cDNA product to produce sequence reads. 
     
     
         4 . The method of  claim 2 , wherein the copies of cDNA product are detected with fluorescence detection. 
     
     
         5 . The method of  claim 1 , wherein said detecting step comprises quantifying said cDNA. 
     
     
         6 . The method of  claim 1 , further comprising introducing an amount of exogenous synthetic RNA, DNA, or mRNA. 
     
     
         7 . The method of  claim 6 , further comprising normalizing the detected cDNA against the exogenous synthetic RNA, DNA, or mRNA. 
     
     
         8 . The method of  claim 1 , further comprising collecting the virion particle from an isolated biological sample. 
     
     
         9 . The method of  claim 8 , wherein the isolated biological sample is a T-cell. 
     
     
         10 . The method of  claim 9 , wherein the T-cell is a CD4+ T cell. 
     
     
         11 . The method of  claim 1 , wherein the latent retrovirus a human immunodefficiency virus (HIV). 
     
     
         12 . A method for detecting a latent retrovirus species, the method comprising the steps of:
 isolating virion RNA in an aqueous droplet surrounded by an immiscible fluid,   exposing the virion RNA in the droplet to at least one primer capable of hybridizing to at least a portion of the virion RNA;   synthesizing a cDNA product from the virion RNA; and   detecting the cDNA product.   
     
     
         13 . The method of  claim 12 , wherein detecting comprises amplifying the cDNA product to produce a plurality of substantially identical copies of the cDNA product. 
     
     
         14 . The method of  claim 13 , further comprising sequencing the substantially identical copies of the cDNA product to produce sequence reads. 
     
     
         15 . The method of  claim 13 , wherein the copies of cDNA product are detected with fluorescence detection. 
     
     
         16 . The method of  claim 12 , wherein said detecting step comprises quantifying said cDNA. 
     
     
         17 . The method of  claim 12 , further comprising introducing an amount of exogenous synthetic RNA, DNA, or mRNA. 
     
     
         18 . The method of  claim 17 , further comprising normalizing the detected cDNA against the exogenous synthetic RNA, DNA, or mRNA. 
     
     
         19 . The method of  claim 12 , further comprising collecting the virion particle from an isolated biological sample. 
     
     
         20 . The method of  claim 19 , wherein the isolated biological sample is a T-cell.