Method and kit for detecting resistance to antiviral drugs

Abstract

Assays and kits for the detection of phenotypic resistance of a retrovirus to reverse transcriptase inhibitor drugs in a biological sample. The assays are based on the direct analysis of the susceptibility of retroviral reverse transcriptase to inhibition by a reverse transcriptase inhibitor drug. The enzymatic activity of the reverse transcriptase is determined by measuring the DNA product produced when an RNA template and a first complementary DNA primer from a suitable region of the encephalomyocarditis virus genome are incubated with a biological sample containing reverse transcriptase in the presence of the drug to which resistance is being determined. The incubation mixture is reacted under conditions whereby the RNA template and the DNA primer will anneal and a DNA strand will be synthesized as an extension from the DNA primer if the reverse transcriptase in the sample is resistant to and not inhibited by the drug. The DNA product is amplified using a second complementary DNA primer from the encephalomyocarditis virus genome and suitable PCR reagents and conditions, and the amplified product detected in accordance with methods known to those skilled in the art. Detection of the amplified DNA indicates resistance to the drug employed in the assay. Detection of relatively greater amounts of amplified DNA when certain drugs are used indicates the presence of multiple nucleoside analog resistant strains or mutations.

1 . A method for the detection of drug resistance of a retrovirus comprising incubating the retrovirus with a reverse transcriptase inhibitor antiviral drug, an RNA template, and a first functionally complementary DNA primer, and 
 detecting the presence or absence of a first DNA product, wherein the detection of the first DNA product indicates resistance to the drug.    
     
     
         2 . The method of  claim 1  wherein the RNA template and the first functionally complementary DNA primer are oligonucleotides that comprise sequence consisting essentially of sequence from a region of the encephalomyocarditis virus genome or its complement having no significant secondary structure and less than 50% G-C content.  
     
     
         3 . The method of  claim 1  wherein the RNA template is an oligonucleotide having a sequence substantially the same as the sequence set forth in SEQ ID NO: 4.  
     
     
         4 . The method of  claim 1  wherein the RNA template is an oligonucleotide having the sequence set forth in SEQ ID NO: 4.  
     
     
         5 . The method of  claim 1  wherein the first complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 2.  
     
     
         6 . The method of  claim 1  wherein the reverse transcriptase inhibitor antiviral drug is selected from the group consisting of lamivudine, 3TC-TP, zalcitabine, ddC-TP, didanosine, ddA-TP, stavudine, d4T-TP, zidovudine, AZT-triphosphate, nevirapine, abacavir, delavirdine, loviride, efavirenz, and adefovir.  
     
     
         7 . The method of  claim 1  wherein the DNA product is detected by hybridization to a detectable hybridization probe, wherein the hybridization probe is an oligonucleotide having the sequence set forth in SEQ ID NO: 3.  
     
     
         8 . The method of  claim 1  further comprising incubating the retrovirus, RNA template and first functionally complementary DNA primer with a second functionally complementary DNA primer comprising sequence corresponding to sequence from the encephalomyocarditis virus genome, wherein the second functionally complementary DNA primer is extended during amplification to form a second DNA product.  
     
     
         9 . The method of  claim 8  wherein the second functinonally complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 1.  
     
     
         10 . The method of  claim 1  further comprising contacting the first DNA product with a second functionally complementary DNA primer comprising sequence consisting essentially of sequence from the encephalomyocarditis virus genome, wherein the second complementary DNA primer is extended to form a second DNA product.  
     
     
         11 . A kit for the detection of antiviral drug resistance of a retrovirus comprising: 
 a) a region of the encephalomyocarditis virus genome having no significant secondary structure and less than 50% G-C content as an RNA template;    b) a first complementary DNA primer for reverse transcriptase, wherein the primer is complementary to a portion of the RNA template;    c) a second complementary DNA primer for amplification, wherein the primer is complementary to a portion of a nucleic acid complementary to the RNA template; and    d) a 5′-triphosphate form of a reverse transcriptase inhibitor antiviral drug to which resistance is being detected.    
     
     
         12 . The kit of  claim 11  wherein the RNA template is an oligonucleotide having the sequence set forth in SEQ ID NO: 4, the first complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 2, the second complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 1; and the reverse transcriptase inhibitor drug is selected from the group consisting of lamivudine, zalcitabine, didanosine, stavudine, zidovudine, AZT-triphosphate, nevirapine, abacavir, delavirdine, loviride, and efavirenz.  
     
     
         13 . The kit of  claim 11  further comprising a hybridization probe for detection of a DNA product, wherein the hybridization probe is an oligonucleotide having the sequence set forth in SEQ ID NO: 3.  
     
     
         14 . A method for the detection of multiple nucleoside analog resistance of a retrovirus comprising: 
 incubating the retrovirus with a reverse transcriptase inhibitor antiviral drug that is a nucleoside analog, an RNA template, and a first functionally complementary DNA primer; and    detecting the relative presence or relative absence of a first DNA product, wherein the detection of the first DNA product indicates resistance to the drug.    
     
     
         15 . The method of  claim 14  wherein the RNA template and first functionally complementary DNA primer are oligonucleotides from a region of the encephalomyocarditis virus genome having no significant secondary structure and less than 50% G-C content.  
     
     
         16 . The method of  claim 14  wherein the nucleoside analog is AZT-triphosphate.  
     
     
         17 . The method of  claim 14  wherein the RNA template is an oligonucleotide having the sequence set forth in SEQ ID NO: 4  
     
     
         18 . The method of  claim 14  wherein the first complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 2.  
     
     
         19 . The method of  claim 14  wherein the first DNA product is detected by hybridization of a detectable hybridization probe, wherein the hybridization probe is an oligonucleotide having the sequence set forth in SEQ ID NO: 3.  
     
     
         20 . The method of  claim 14  further comprising incubating the retrovirus, RNA template and first complementary DNA primer with a second complementary DNA primer consisting essentially of sequence from the encephalomyocarditis virus genome, wherein the second complementary DNA primer is extended during amplification to form a second DNA product.  
     
     
         21 . The method of  20  wherein the second complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 1.  
     
     
         22 . The method of  claim 20  wherein a DNA product selected from the group consisting of the first DNA product and the second DNA product is detected by hybridizing a detectable oligonucleotide to the DNA product.  
     
     
         23 . The method of  claim 22  wherein the detectable oligonucleotide is an internal EMCV-specific probe.  
     
     
         24 . The method of  claim 23  wherein the detectable oligonucleotide comprises sequence set forth in SEQ ID NO: 3.  
     
     
         25 . The method of  claim 24  wherein the detectable oligonucleotide is an oligonucleotide consisting of the sequence of SEQ ID NO: 3.  
     
     
         26 . A method for characterizing strains of HIV-1 or HIV-2 as being MNR mutants and as being distinct from wild-type or classical reverse transcriptase mutant strains, comprising: 
 incubating a strain of HIV-1 with a 5′-triphosphate version of a reverse transcriptase inhibitor antiviral drug, an RNA template, and a first complementary DNA primer, and    detecting the relative presence or relative absence of a first DNA product, wherein the detection of the first DNA product indicates that the strain of HIV-1 is an MNR mutant strain and is neither a wild-type nor a classical reverse transcriptase mutant strain.    
     
     
         27 . The method of  claim 26  wherein the RNA template and first functionally complementary DNA primer are oligonucleotides that comprise sequence consisting essentially of sequence from a region of the encephalomyocarditis virus genome having no significant secondary structure and less than 50% G-C content.  
     
     
         28 . The method of  claim 27  wherein the RNA template is an oligonucleotide having a sequence substantially the same as the sequence set forth in SEQ ID NO: 4.  
     
     
         29 . The method of  claim 27  wherein the RNA template is an oligonucleotide having the sequence set forth in SEQ ID NO: 4.  
     
     
         30 . The method of  claim 27  wherein the first complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 2.  
     
     
         31 . The method of  claim 27  wherein the reverse transcriptase inhibitor is AZT-triphosphate.  
     
     
         32 . The method of  claim 27  wherein the DNA product is detected by hybridization to a detectable hybridization probe, wherein the hybridization probe is an oligonucleotide having the sequence set forth in SEQ ID NO: 3.  
     
     
         33 . The method of  claim 26  further comprising incubating the retrovirus, RNA template and first complementary DNA primer with a second complementary DNA primer comprising sequence corresponding to sequence from the encephalomyocarditis virus genome, wherein the second complementary DNA primer is extended during amplification to form a second DNA product.  
     
     
         34 . The method of  claim 30  wherein the second complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 1.  
     
     
         35 . The method of  claim 26  further comprising contacting the first DNA product with a second functionally complementary DNA primer comprising sequence consisting essentially of sequence from the encephalomyocarditis virus genome, wherein the second functionally complementary DNA primer is extended to form a second DNA product.  
     
     
         36 . A method for identifying the presence of retrovirus strains with mutations that confer multiple nucleoside analog resistance, comprising: 
 incubating a sample suspected of containing retrovirus with a reverse transcriptase inhibitor antiviral drug wherein the drug is a nucleoside analog, an RNA template; and    detecting the relative presence or relative absence of a first DNA product, wherein the detection of the first DNA product indicates the presence of retrovirus strains having multiple nucleoside analog resistance.    
     
     
         37 . The method of  claim 36  wherein the RNA template and first functionally complementary DNA primer are oligonucleotides that comprise sequence corresponding to sequence from a region of the encephalomyocarditis virus genome having no significant secondary structure and less than 50% G-C content.  
     
     
         38 . The method of  claim 36  wherein detection or absence of a first DNA product is monitored as a function of the degree of inhibition of first DNA product formed and wherein inhibition of less than 90% indicates the presence of MNR strains.  
     
     
         39 . The method of  claim 36  wherein the RNA template is an oligonucleotide having a sequence substantially the same as the sequence set forth in SEQ ID NO: 4.  
     
     
         40 . The method of  claim 36  wherein the RNA template is an oligonucleotide having the sequence set forth in SEQ ID NO: 4.  
     
     
         41 . The method of  claim 36  wherein the first complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 2.  
     
     
         42 . The method of  claim 36  wherein the reverse transcriptase inhibitor is AZT-triphosphate.  
     
     
         43 . The method of  claim 42  wherein 1 μM AZT triphosphate is used as the reverse transcriptase inhibitor drug.  
     
     
         44 . The method of  claim 36  wherein the DNA product is detected by hybridization to a detectable hybridization probe, wherein the hybridization probe is an oligonucleotide having the sequence set forth in SEQ ID NO: 3.  
     
     
         45 . The method of  claim 36  further comprising incubating the retrovirus, RNA template and first functionally complementary DNA primer with a second functionally complementary DNA primer comprising sequence corresponding to sequence from the encephalomyocarditis virus genome, wherein the second complementary DNA primer is extended during amplification to form a second DNA product.  
     
     
         46 . The method of  claim 45  wherein the second functionally complementary DNA primer is an oligonucleotide having the sequence set forth in SEQ ID NO: 1.