System and method for detection of HIV tropism variants

Abstract

An embodiment of a method for detecting low frequency occurrence of one or more HIV sequence variants associated with drug resistance is described that comprises the steps of: generating cDNA species from each RNA molecule in an HIV sample population; amplifying at least one first amplicon from the cDNA species, wherein each first amplicon comprises a plurality of amplified copies and is amplified with a pair of nucleic acid primers that define a locus of the first amplicon; clonally amplifying the amplified copies of the first amplicons to produce a plurality of second amplicons wherein a plurality of the second amplicons comprise an immobilized population of substantially identical copies from one of the amplified copies of first amplicons; determining a nucleic acid sequence composition of the substantially identical copies from at least 100 of the immobilized populations in parallel on a single substrate; and detecting one or more sequence variants that occur at a frequency of 5% or less in the nucleic acid sequence composition of the at least 100 immobilized populations; and correlating the detected sequence variants with variation associated with HIV tropism.

What is claimed is: 
     
       1. A nucleic acid sequencing kit for detecting a low frequency occurrence of one or more HIV Tropism sequence variants, comprising:
 a plurality of nucleic acid primers adapted to clonally amplify by PCR and sequence an HIV V3 region from a plurality of HIV clades comprising clade A, clade B, clade C, clade D, and clade G, wherein the nucleic acid primers comprise a V3-1F primer (SEQ ID NO: 5 or SEQ ID NO: 1); a V3-1R primer (SEQ ID NO: 6); and a V3-2F primer (SEQ ID NO: 7 or SEQ ID NO: 3). 
 
     
     
       2. The kit of  claim 1 , further comprising: a V3-1R primer (SEQ ID NO: 2) adapted to amplify a plurality of cDNA species from a plurality of RNA molecules in an HIV sample population derived from a single patient. 
     
     
       3. The method of  claim 2 , wherein:
 the plurality of cDNA species have overlapping sequence composition. 
 
     
     
       4. The kit of  claim 1 , wherein:
 the plurality of primers target regions of low mutation frequency. 
 
     
     
       5. The kit of  claim 1 , wherein:
 the plurality of nucleic acid primers comprise pairs adapted to target a locus comprising the V3 region of HIV. 
 
     
     
       6. The kit of  claim 4 , wherein:
 the V3 region is associated with an HIV envelope region. 
 
     
     
       7. The kit of  claim 1 , further comprising:
 a single substrate comprising a plurality of reaction sites. 
 
     
     
       8. The kit of  claim 1  wherein:
 the plurality of HIV clades further comprise clade F, clade H, clade J, and clade K. 
 
     
     
       9. The kit of claim 1 wherein the nucleic acid primers comprise a V3-1F primer (SEQ ID NO: 1) and a V3-2F primer (SEQ ID NO: 3) and further comprising a V3-2R primer (SEQ ID NO:4). 
     
     
       10. The kit of claim 1 wherein the nucleic acid primers comprise a V3-1F primer (SEQ ID NO: 5); a V3-1R primer (SEQ ID NO: 6); and a V3-2F primer (SEQ ID NO: 7).