SYSTEM AND METHOD FOR DETECTION OF HIV DRUG RESISTANT VARIANTS

• US 20100203497A1
• Filed: 03/14/2008
• Published: 08/12/2010
• Est. Priority Date: 03/16/2007
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Lubeski, Christine, Simons, Jan Fredrik, Simen, Birgitte Binderup
• Granted Patent: US 8,617,816 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

In one embodiment of the invention a method for detecting low frequency occurrence of one or more HIV sequence variants associated with drug resistance is describe that comprises generating cDNA species from RNA molecules in an HIV sample population; amplifying first amplicons from the cDNA species, wherein each amplicon comprises amplified copies and is amplified with a pair of nucleic acid primers that define a locus; clonally amplifying the amplified copies of the first amplicons to produce second amplicons that comprise an immobilized population of substantially identical copies from one of the amplified copies of first amplicons; determining a nucleic acid sequence composition from at least 100 of the immobilized populations in parallel on a single instrument; 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 drug resistance.

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 each RNA molecule in an HIV sample population;

(b) amplifying a plurality of first amplicons 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;

(b) 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;

(c) determining a nucleic acid sequence composition of the substantially identical copies from at least 100 of the immobilized populations in parallel on a single instrument; and

(d) 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

(e) correlating the detected sequence variants with variation associated with HIV drug resistance.

2. The method of claim 1, wherein:

the variation associated with HIV drug resistance is known to be associated with a particular drug class or drug.

3. The method of claim 2, wherein:

the HIV drug class is selected from the group consisting of protease inhibitors, nucleotide/nucleoside reverse transcriptase inhibitors, and non-nucleoside reverse transcriptase inhibitors.

4. The method of claim 1, wherein:

the plurality of cDNA species includes 3 cDNA species.

5. The method of claim 1, wherein:

each of the 3 cDNA species have overlapping sequence composition with at least one neighboring cDNA species.

6. The method of claim 1, wherein:

the HIV sample population is derived from a single patient.

7. The method of claim 1, wherein:

the single patient is drug naï

ve.

8. The method of claim 1, wherein:

the single patient was previously exposed to HIV anti-retorviral drug therapy.

9. The method of claim 1, wherein:

the plurality of first amplicons comprises 8 amplicons.

10. The method of claim 1, wherein:

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

11. The method of claim 1, wherein:

the pair of primers for the first amplicons are specific to an HIV Glade selected from the group consisting of Glade B, and Glade C.

12. The method of claim 11, wherein:

the pair of primers for the first amplicons specific to Glade B comprise a group of primer pairs selected from the group consisting of B-ACF-1 (SeqID No;

     39) and B-AR (SeqID No;

     36);

B-ACF-2 (SeqID No;

     40) and B-AR (SeqID No;

     36);

B-BF (SeqID No;

     35) and B-BR (SeqID No;

     38);

B-ACF-1 (SeqID No;

     39) and B-CR (SeqID No;

     37);

B-ACF-2 (SeqID No;

     40) and B-CR (SeqID No;

     37);

B-1F (SeqID No;

     42) and B-1R-1 (SeqID No;

     44);

B-1F (SeqID No;

     42) and B-1R-2 (SeqID No;

     45);

B-2F (SeqID No;

     43) and B-2R (SeqID No;

     49);

B-3F (SeqID No;

     50) and B-3R (SeqID No;

     51);

B-4F (SeqID No;

     41) and B-4R-1 (SeqID No;

     46);

B-4F (SeqID No;

     41) and B-4R-2 (SeqID No;

     47);

B-5F (SeqID No;

     48) and B-5R-1 (SeqID No;

     52); and

B-5F (SeqID No;

     48) and B-5R-2 (SeqID No;

     53).

13. The method of claim 11, wherein:

the pair of primers for the first amplicons specific to Glade C comprise a group of primer pairs selected from the group consisting of C-ACF-1 (SeqID No;

     54) and C-AR-1 (SeqID No;

     58);

C-ACF-1 (SeqID No;

     54) and C-AR-2 (SeqID No;

     59);

C-ACF-2 (SeqID No;

     55) and C-AR-1 (SeqID No;

     58);

C-ACF-2 (SeqID No;

     55) and C-AR-2 (SeqID No;

     59);

C-BF-1 (SeqID No;

     56) and C-BR (SeqID No;

     61);

C-BF-2 (SeqID No;

     57) mix and C-BR (SeqID No;

     61);

C-ACF-1 (SeqID No;

     54) and C-CR (SeqID No;

     60);

C-ACF-2 (SeqID No;

     55) and C-CR (SeqID No;

     60);

C-1F-1 (SeqID No;

     63) and C-1R-1 (SeqID No;

     65);

C-1F-1 (SeqID No;

     63) and C-1R-2 (SeqID No;

     66);

C-1F-2 (SeqID No;

     64) and C-1R-1 (SeqID No;

     65);

C-1F-2 (SeqID No;

     64) and C-1R-2 (SeqID No;

     66);

C-2F-1 (SeqID No;

     67) and C-2R (SeqID No;

     73);

C-2F-2 (SeqID No;

     68) and C-2R (SeqID No;

     73);

C-3F-1 (SeqID No;

     74) and C-3R (SeqID No;

     78);

C-3F-2 (SeqID No;

     75) and C-3R (SeqID No;

     78);

C-3F-3 (SeqID No;

     76) and C-3R (SeqID No;

     78);

C-3F-4 (SeqID No;

     77) and C-3R (SeqID No;

     78);

C-4F (SeqID No;

     62) and C-4R-1 (SeqID No;

     69);

C-4F (SeqID No;

     62) and C-4R-2 (SeqID No;

     70);

C-5F-1 (SeqID No;

     71) and C-5R (SeqID No;

     79); and

C-5F-2 (SeqID No;

     72) and C-5R (SeqID No;

     79).

14. The method of claim 1, wherein:

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

15. The method of claim 1, wherein:

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

16. The method of claim 1, wherein:

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

17. The method of claim 1, wherein:

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

18. 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 1.25% or less.

19. 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 0.050% or less.

20. 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.

21. The method of claim 1 wherein:

the single instrument comprises a single substrate comprising a plurality of reaction sites.

22. The method of claim 1 wherein:

the single instrument comprises a single detection device capable of detecting signals generated from a plurality of sequencing reactions.

23. A kit for performing the method of claim 1, Comprising:

one or more pairs of primers specific to HIV Glade B selected from the group consisting of B-ACF-1 (SeqID No;

     39) and B-AR (SeqID No;

     36);

B-ACF-2 (SeqID No;

     40) and B-AR (SeqID No;

     36);

B-BF (SeqID No;

     35) and B-BR (SeqID No;

     38);

B-ACF-1 (SeqID No;

     39) and B-CR (SeqID No;

     37);

B-ACF-2 (SeqID No;

     40) and B-CR (SeqID No;

     37);

B-1F (SeqID No;

     42) and B-1R-1 (SeqID No;

     44);

B-1F (SeqID No;

     42) and B-1R-2 (SeqID No;

     45);

B-2F (SeqID No;

     43) and B-2R (SeqID No;

     49);

B-3F (SeqID No;

     50) and B-3R (SeqID No;

     51);

B-4F (SeqID No;

     41) and B-4R-1 (SeqID No;

     46);

B-4F (SeqID No;

     41) and B-4R-2 (SeqID No;

     47);

B-5F (SeqID No;

     48) and B-5R-1 (SeqID No;

     52); and

B-5F (SeqID No;

     48) and B-5R-2 (SeqID No;

     53).

24. A kit for performing the method of claim 1, comprising:

one or more pairs of primers specific to HIV Glade C selected from the group consisting of C-ACF-1 (SeqID No;

     54) and C-AR-1 (SeqID No;

     58);

C-ACF-1 (SeqID No;

     54) and C-AR-2 (SeqID No;

     59);

C-ACF-2 (SeqID No;

     55) and C-AR-1 (SeqID No;

     58);

C-ACF-2 (SeqID No;

     55) and C-AR-2 (SeqID No;

     59);

C-BF-1 (SeqID No;

     56) and C-BR (SeqID No;

     61);

C-BF-2 (SeqID No;

     57) mix and C-BR (SeqID No;

     61);

C-ACF-1 (SeqID No;

     54) and C-CR (SeqID No;

     60);

C-ACF-2 (SeqID No;

     55) and C-CR (SeqID No;

     60);

C-1F-1 (SeqID No;

     63) and C-1R-1 (SeqID No;

     65);

C-1F-1 (SeqID No;

     63) and C-1R-2 (SeqID No;

     66);

C-1F-2 (SeqID No;

     64) and C-1R-1 (SeqID No;

     65);

C-1F-2 (SeqID No;

     64) and C-1R-2 (SeqID No;

     66);

C-2F-1 (SeqID No;

     67) and C-2R (SeqID No;

     73);

C-2F-2 (SeqID No;

     68) and C-2R (SeqID No;

     73);

C-3F-1 (SeqID No;

     74) and C-3R (SeqID No;

     78);

C-3F-2 (SeqID No;

     75) and C-3R (SeqID No;

     78);

C-3F-3 (SeqID No;

     76) and C-3R (SeqID No;

     78);

C-3F-4 (SeqID No;

     77) and C-3R (SeqID No;

     78);

C-4F (SeqID No;

     62) and C-4R-1 (SeqID No;

     69);

C-4F (SeqID No;

     62) and C-4R-2 (SeqID No;

     70);

C-5F-1 (SeqID No;

     71) and C-5R (SeqID No;

     79); and

C-5F-2 (SeqID No;

     72) and C-5R (SeqID No;

     79).