Assay for detecting and quantifying HIV-1

Abstract

Compositions, methods and kits for detecting HIV-1 nucleic acids using nucleic acid amplification. Particularly described are oligonucleotides that are useful as hybridization probes and amplification primers for detecting very low levels of HIV-1 nucleic acids by real-time monitoring of amplicon production. The invented assays are characterized by high levels of precision in the quantitation of HIV-1 targets at low copy numbers, and by accurate detection of different HIV-1 subtypes, including M group and O group variants.

1 . A reaction mixture for amplifying either HIV-1 M group nucleic acids or HIV-1 O group nucleic acids, comprising: 
 (a) a first amplification primer comprising a first primer target-hybridizing sequence that independently hybridizes to a first strand of HIV-1 M group nucleic acids and a first strand of HIV-1 O group nucleic acids; and    (b) a second amplification primer comprising a second primer target-hybridizing sequence that hybridizes to an enzymatic extension product of said first amplification primer using as a template either said first strand of HIV-1 M group nucleic acids or said first strand of HIV-1 O group nucleic acids, said second primer target-hybridizing sequence consisting essentially of SEQ ID NO:33.    
     
     
         2 . The reaction mixture of  claim 1 , wherein said second primer target-hybridizing sequence consists essentially of SEQ ID NO:2.  
     
     
         3 . The reaction mixture of  claim 2 , wherein said first primer target-hybridizing sequence consists essentially of SEQ ID NO:13.  
     
     
         4 . The reaction mixture of  claim 2 , wherein said first primer target-hybridizing sequence consists essentially of SEQ ID NO:15.  
     
     
         5 . The reaction mixture of  claim 1 , wherein said second primer target-hybridizing sequence consists essentially of SEQ ID NO:5.  
     
     
         6 . The reaction mixture of  claim 5 , wherein said first primer target-hybridizing sequence consists essentially of SEQ ID NO:15.  
     
     
         7 . The reaction mixture of  claim 1 , further comprising a hybridization probe.  
     
     
         8 . The reaction mixture of  claim 7 , wherein said hybridization probe is selected from the group consisting of a molecular beacon hybridization probe and a molecular torch hybridization probe.  
     
     
         9 . The reaction mixture of  claim 8 , wherein no more than two primers and a single probe are used for amplifying and detecting the HIV-1 M group nucleic acids or the HIV-1 O group nucleic acids.  
     
     
         10 . A method of quantifying the combined amount of an HIV-1 M group nucleic acid and an HIV-1 O group nucleic acid that may be present in a biological sample, comprising the steps of: 
 combining in a single reaction vessel said biological sample, a first amplification primer, a second amplification primer, and a hybridization probe;    amplifying with substantially equal efficiency any of the HIV-1 M group nucleic acid and the HIV-1 O group nucleic acid present in the biological sample using an in vitro amplification reaction that comprises enzymatic extension of said first amplification primer using a first strand of said HIV-1 M group nucleic acid or said HIV-1 O group nucleic acid as a first template to create a first primer extension product, and enzymatic extension of said second amplification primer using said first primer extension product as a second template, 
 whereby there are produced HIV-1 M group amplicons if the biological sample contained said HIV-1 M group nucleic acid, and HIV-1 O group amplicons if the biological sample contained said HIV-1 O group nucleic acid;  
   monitoring amplicon production in said in vitro amplification reaction as a function of time by a process comprising detection of a signal from said hybridization probe, whereby time-dependent quantitative data is obtained; and    quantifying the combined amount of said HIV-1 M group nucleic acid and said HIV-1 O group nucleic acid present in the biological sample using the time-dependent quantitative data obtained in the monitoring step, 
 wherein neither said first amplification primer nor said second amplification primer is fully complementary to said HIV-1 M group nucleic acid or the complement thereof, or to said HIV-1 O group nucleic acid or the complement thereof, and  
 wherein said hybridization probe hybridizes to both HIV-1 M group amplicons and HIV-1 O group amplicons.  
   
     
     
         11 . The method of  claim 10 , wherein said in vitro amplification reaction is an isothermal in vitro amplification reaction.  
     
     
         12 . The method of  claim 11 , wherein said isothermal in vitro amplification reaction is a transcription associated amplification reaction selected from the group consisting of a TMA reaction and a NASBA reaction.  
     
     
         13 . The method of  claim 10 , wherein the signal detected in the monitoring step is a fluorescent signal.  
     
     
         14 . The method of  claim 13 , where said hybridization probe is a molecular torch hybridization probe.  
     
     
         15 . The method of  claim 14 , wherein said first amplification primer comprises a first primer target-hybridizing sequence consisting essentially of SEQ ID NO:15.  
     
     
         16 . The method of  claim 15 , wherein said second amplification primer comprises a second primer target-hybridizing sequence consisting essentially of SEQ ID NO:5.  
     
     
         17 . The method of  claim 10 , wherein no more than two primers and a single probe are used for amplifying and detecting both said HIV-1 M group nucleic acid and said HIV-1 O group nucleic acid.  
     
     
         18 . The method of  claim 17 , wherein said in vitro amplification reaction is an isothermal in vitro amplification reaction.  
     
     
         19 . The method of  claim 17 , wherein the quantifying step comprises comparing a quantitative result with no more than a single standard curve.  
     
     
         20 . A method of establishing a point on a standard curve that can be used for quantifying HIV-1 M group nucleic acids and HIV-1 O group nucleic acids in a single reaction, comprising the steps of: 
 providing a known amount of an HIV-1 standard;    amplifying in an in vitro amplification reaction said HIV-1 standard using a first primer and a second primer in the presence of a hybridization probe to produce HIV-1 standard amplicons, 
 wherein said amplification reaction amplifies HIV-1 M group nucleic acids and HIV-1 O group nucleic acids with substantially equal efficiency;  
   monitoring production of HIV-1 standard amplicons synthesized in said in vitro amplification reaction as a function of time by a process comprising detection of a signal from said hybridization probe, whereby quantitative data is obtained; and    establishing from the quantitative data said point on said standard curve.    
     
     
         21 . The method of  claim 20 , wherein said first amplification primer comprises a first primer target-hybridizing sequence that independently hybridizes to a first strand of HIV-1 M group nucleic acids and a first strand of HIV-1 O group nucleic acids, 
 wherein said second amplification primer comprises a second primer target-hybridizing sequence that hybridizes to an enzymatic extension product of said first amplification primer using as a template either said first strand of HIV-1 M group nucleic acids or said first strand of HIV-1 O group nucleic acids,    wherein neither said first primer target-hybridizing sequence nor said second primer target-hybridizing sequence is fully complementary to HIV-1 M group or HIV-1 O group nucleic acids or the complements thereof, and    wherein said hybridization probe hybridizes either to HIV-1 M group nucleic acids and HIV-1 O group nucleic acids, or to the complements thereof.    
     
     
         22 . The method of  claim 21 , wherein said hybridization probe is a molecular torch.  
     
     
         23 . The method of  claim 21 , wherein said HIV-1 standard is an HIV-1 M group nucleic acid standard.  
     
     
         24 . The method of  claim 23 , further comprising a step for using the standard curve to quantify an HIV-1 O group nucleic acid contained in a biological sample.  
     
     
         25 . The method of  claim 20 , wherein said HIV-1 standard is an HIV-1 O group nucleic acid standard.  
     
     
         26 . The method of  claim 25 , further comprising a step for using the standard curve to quantify an HIV-1 M group nucleic acid contained in a biological sample.  
     
     
         27 . The method of  claim 21 , wherein said in vitro amplification reaction in the amplifying step is an isothermal in vitro amplification reaction.  
     
     
         28 . The method of  claim 27 , wherein said isothermal in vitro amplification reaction is a transcription associated amplification reaction selected from the group consisting of a TMA reaction and a NASBA reaction.  
     
     
         29 . The method of  claim 28 , wherein the step for monitoring comprises measuring a fluorescent signal.  
     
     
         30 . A method of preparing a reaction mixture for amplifying either or both of HIV-1 M group nucleic acids and HIV-1 O group nucleic acids, comprising: 
 (a) selecting a first amplification primer comprising a sequence that independently hybridizes to a first strand of either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids;    (b) selecting a second amplification primer comprising a sequence that hybridizes to enzymatic extension products of said first amplification primer using said first strand of either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids as a template;    (c) selecting a hybridization probe that hybridizes to amplicons synthesized by the use of said first and said second amplification primers, 
 wherein neither said first primer target-hybridizing sequence nor said second primer target-hybridizing sequence is fully complementary to said HIV-1 M group or HIV-1 O group nucleic acids or the complements thereof, and  
 wherein said first amplification primer, said second amplification primer, and said hybridization probe are further selected to amplify in an in vitro amplification reaction HIV-1 M group nucleic acids and HIV-1 O group nucleic acids with substantially equal efficiencies; and  
   (d) combining in a single reaction vessel said first amplification primer, said second amplification primer, and said hybridization probe.    
     
     
         31 . The method of  claim 30 , wherein said reaction mixture comprises no more than two primers and a single hybridization probe for amplifying and detecting the HIV-1 M group nucleic acids and HIV-1 O group nucleic acids.  
     
     
         32 . The method of  claim 31 , wherein said in vitro amplification reaction is an isothermal in vitro amplification reaction.  
     
     
         33 . The method of  claim 32 , wherein said isothermal in vitro amplification reaction is a transcription associated amplification reaction selected from the group consisting of a TMA reaction and a NASBA reaction.  
     
     
         34 . A composition for amplifying HIV-1 M group target nucleic acids and HIV-1 O group target nucleic acids, comprising: 
 a first amplification primer comprising a first primer target-hybridizing sequence that independently hybridizes to a first strand of HIV-1 M group target nucleic acids and a first strand of HIV-1 O group target nucleic acids;    a second amplification primer comprising a second primer target-hybridizing sequence that hybridizes to enzymatic extension products of said first amplification primer using said first strand of either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids as a template, 
 wherein neither said first primer target-hybridizing sequence nor said second primer target-hybridizing sequence is fully complementary to said HIV-1 M group or HIV-1 O group target nucleic acids or the complements thereof.  
   
     
     
         35 . The composition of  claim 34 , further comprising a hybridization probe that hybridizes to an amplification product produced in an in vitro amplification reaction by the combined activity of said first and second amplification primers using as a template either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids.  
     
     
         36 . The composition of  claim 35 , wherein said composition amplifies HIV-1 M group target nucleic acids and HIV-1 O group target nucleic acids in said in vitro nucleic acid amplification reaction with substantially equal efficiency.  
     
     
         37 . The composition of  claim 36 , wherein said first primer target-hybridizing sequence consists essentially of SEQ ID NO:15, and wherein said second primer target-hybridizing sequence consists essentially of SEQ ID NO:5.  
     
     
         38 . The composition of  claim 36 , wherein said hybridization probe is a molecular torch.  
     
     
         39 . The composition of  claim 35 , wherein said hybridization probe is selected from the group consisting of a molecular beacon and a molecular torch.  
     
     
         40 . The composition of  claim 39 , wherein said hybridization probe is the molecular torch.  
     
     
         41 . The composition of  claim 34 , wherein said second primer target-hybridizing sequence consists essentially of SEQ ID NO:5.  
     
     
         42 . The composition of  claim 41 , wherein said first primer target-hybridizing sequence consists essentially of SEQ ID NO:15.  
     
     
         43 . The composition of  claim 35 , wherein said first primer target-hybridizing sequence consists essentially of SEQ ID NO:15, and wherein said second primer target-hybridizing sequence consists essentially of SEQ ID NO:5.  
     
     
         44 . The composition of  claim 43 , wherein said hybridization probe is a molecular torch.  
     
     
         45 . A reaction mixture for amplifying either HIV-1 M group nucleic acids or HIV-1 O group nucleic acids, comprising: 
 (a) a first amplification primer comprising a first primer target-hybridizing sequence that independently hybridizes to a first strand of HIV-1 M group nucleic acids and a first strand of HIV-1 O group nucleic acids;    (b) a second amplification primer comprising a second primer target-hybridizing sequence that hybridizes to an enzymatic extension product of said first amplification primer using as a template either said first strand of HIV-1 M group nucleic acids or said first strand of HIV-1 O group nucleic acids; and    (c) a molecular torch hybridization probe that hybridizes to an amplicon synthesized by the combined activity of said first amplification primer and said second amplification primer, 
 wherein neither said first primer target-hybridizing sequence nor said second primer target-hybridizing sequence is fully complementary to said HIV-1 M group or HIV-1 O group nucleic acids or the complement thereof, and  
 wherein said HIV-1 M group nucleic acids and HIV-1 O group nucleic acids amplify in the reaction mixture with substantially equal efficiency.