System and method for detection of HIV tropism variants

• US 7,888,034 B2
• Filed: 06/17/2009
• Issued: 02/15/2011
• Est. Priority Date: 07/01/2008
• Status: Expired due to Fees

• 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; Suryaprabha
• Application Number:
  US12/456,528
• Publication Number:
  US 20100003687A1
• Time in Patent Office: 608 Days
• Field of Search: 435/6, 435/91.2, 536/24.33
• US Class Current: 435/5
• 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 method for detecting low frequency occurrence of one or more HIV sequence variants associated with drug resistance comprising the steps of:

(a) generating a plurality of cDNA species from a plurality of RNA molecules in an HIV sample population derived from a single patient, wherein each of the cDNA species is generated using a pair of nucleic acid primers that target an HIV V3 region of an HIV clade selected from the group consisting of clade A, clade B, clade C, clade D, and clade G, and wherein the pair of nucleic acid primers are selected from the group consisting of V3-1F (SEQ ID NO;

     1) and V3-1R (SEQ ID NO;

     2);

V3-2F (SEQ ID NO;

     3) and V3-2R (SEQ ID NO;

     4);

V3-1F (SEQ ID NO;

     1) and V3-2R (SEQ ID NO;

     4); and

V3-2F (SEQ ID NO;

     3) and V3-1R (SEQ ID NO;

     2);

(b) amplifying at least one first amplicon from the cDNA species by PCR, wherein each first amplicon comprises a plurality of amplified copies and is amplified with the pair of nucleic acid primers that define a locus of the first amplicon;

(c) clonally amplifying the amplified copies of the first amplicons by emulsion PCR 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;

(d) 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

(e) 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

(f) correlating the detected sequence variants with variation associated with HIV tropism.

2. The method of claim 1, wherein:

the variation associated with HIV tropism is known to be associated with a coreceptor.

3. The method of claim 2, wherein:

the HIV coreceptor is selected from the group consisting of CCR5 and CXCR4.

4. The method of claim 1, wherein:

the step of generating cDNA species produces 2 cDNA species.

5. The method of claim 4, wherein:

the 2 cDNA species have overlapping sequence composition.

6. The method of claim 1, wherein:

the plurality of first amplicons comprises 2 amplicons.

7. The method of claim 1, wherein:

the pair of primers for the first amplicons target regions of low mutation frequency.

8. The method of claim 1, wherein:

the locus of the first amplicon includes a region of HIV associated with V3 region.

9. The method of claim 8, wherein:

the V3 region is associated with an HIV envelope region.

10. The method of claim 1, wherein:

the second amplicons are amplified using a pair of general primers.

11. The method of claim 1, wherein:

each sequence variant is detected at a 99% confidence level.

12. The method of claim 1 wherein:

the nucleic acid composition of the substantially identical copies from at least 400 immobilized populations is determined and each of the detected sequence variants occur at a frequency of 1.85% or less.

13. The method of claim 1 wherein:

the nucleic acid composition of the substantially identical copies from at least 10000 immobilized populations is determined and each of the detected sequence variants occur at a frequency of 0.74% or less.

14. The method of claim 1 wherein:

the nucleic acid composition of the substantially identical copies from at least 200000 immobilized populations is determined and each of the detected sequence variants occur at a frequency of 0.003% or less.

15. The method of claim 1 wherein:

the step of detecting employs an instrument comprising a single detection device capable of detecting signals generated from a plurality of sequencing reactions on the single substrate.