Simultaneous quantification of nucleic acids in diseased cells

Abstract

This invention pertains to a novel method to screen the efficacy of various anti-viral, and especially anti-HIV and HCV agents by using a novel real-time polymerase chain reaction technique. This method can also be applied to toxicity screening, especially of mitochondrial toxicity of these compounds as well.

We claim:  
     
         1 . A process for identifying a compound which inhibits viral replication that includes contacting nucleic acids from a virus infected host with an amplification reaction mixture that contains at least two primers and/or probes that provide detectable signals during a polymerase chain reaction, wherein 
 the first primer and/or probe provides a detectable signal on the occurrence of the transcription of viral nucleic acids; and    the second primer and/or probe provides a second detectable signal on the occurrence of the transcription of host nucleic acids.    
     
     
         2 . The process of  claim 1 , wherein the host nucleic acid is nuclear nucleic acid.  
     
     
         3 . The process of  claim 1 , wherein the host nucleic acid is mitochondrial nucleic acid.  
     
     
         4 . The process of  claim 3 , wherein the mitochondrial nucleic acid is mitochondrial DNA.  
     
     
         5 . The process of  claim 3 , wherein the mitochondrial nucleic acid is mitochondrial RNA.  
     
     
         6 . The process of  claim 1 , wherein the viral nucleic acid is a non-coding sequence.  
     
     
         7 . The process of  claim 6 , wherein the non-coding sequence is a 5′-non-coding sequence.  
     
     
         8 . The process of  claim 6 , wherein the non-coding sequence is a 3′-non-coding sequence.  
     
     
         9 . The process of  claim 6 , wherein the non-coding sequence is an intron.  
     
     
         10 . The process of  claim 6 , wherein the non-coding sequence is from β-actin.  
     
     
         11 . The process of  claim 6 , wherein the non-coding sequence is from GAPDH.  
     
     
         12 . The process of  claim 1 , wherein the viral nucleic acid is a coding sequence.  
     
     
         13 . The process of  claim 12 , wherein the coding sequence is from HIV.  
     
     
         14 . The process of  claim 12 , wherein the coding sequence is from HBV.  
     
     
         15 . The process of  claim 12 , wherein the coding sequence is from HCV.  
     
     
         16 . The process of  claim 12 , wherein the coding sequence is from BVDV.  
     
     
         17 . The process of  claim 12 , wherein the coding sequence is from West Nile Virus.  
     
     
         18 . The process of  claim 12 , wherein the coding sequence is from herpes.  
     
     
         19 . The process of  claim 12 , wherein the coding sequence is from influenza.  
     
     
         20 . The process of  claim 12 , wherein the coding sequence is from RSV.  
     
     
         21 . The process of  claim 12 , wherein the coding sequence is from EBV.  
     
     
         22 . The process of  claim 12 , wherein the coding sequence is from CMV.  
     
     
         23 . A process for assessing the toxicity of a compound that includes contacting nucleic acids from a host with an amplification reaction mixture that contains at least two primers and/or probes that provide detectable signals during a polymerase chain reaction, wherein 
 the first primer and/or probe provides a detectable signal on the occurrence on the transcription of host mitochondrial nucleic acids; and    the second primer and/or probe provides a second detectable signal on the occurrence on the transcription of host nuclear nucleic acid.    
     
     
         24 . The process of  claim 23 , wherein the host mitochondrial nucleic acid is mitochondrial DNA.  
     
     
         25 . The process of  claim 23 , wherein the host mitochondrial nucleic acid is mitochondrial RNA.  
     
     
         26 . The process of  claim 23 , wherein the host mitochondrial nucleic acid is a non-coding sequence.  
     
     
         27 . The process of  claim 26 , wherein the non-coding sequence is a 5′-non-coding sequence.  
     
     
         28 . The process of  claim 26 , wherein the non-coding sequence is a 3′-non-coding sequence.  
     
     
         29 . The process of  claim 26 , wherein the non-coding sequence is an intron.  
     
     
         30 . The process of  claim 26 , wherein the non-coding sequence is from β-actin.  
     
     
         31 . The process of  claim 26 , wherein the non-coding sequence is from GAPDH.  
     
     
         32 . The process of  claim 23 , wherein the host mitochondrial nucleic acid is a coding sequence.