System and method for detection of HIV tropism variants

• US 8,344,123 B2
• Filed: 12/16/2010
• Issued: 01/01/2013
• Est. Priority Date: 07/01/2008
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Simen, Birgitte Binderup, St. John, Elizabeth Patricia
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US12/970,036
• Publication Number:
  US 20110165556A1
• Time in Patent Office: 747 Days
• Field of Search: None
• US Class Current: 536/24.33
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2549/119 using nested primers

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.

Claims

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);

a and V3-1R primer (SEQ ID NO;

     6); and

a V3-2F primer (SEQ ID NO;

     7).

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.