Comparison of capillary electrophoresis sequencing with the new CEQ 2000 DNA Analysis System to conventional gel based systems for HIV drug resistance analysis.
To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an 'in house' protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and > or =97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was approximately 49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis.
Merel
P
P
Laboratoire de Virologie, Centre Hospitalier Régional et Université Victor Ségalen, Bordeaux, France. patrick.merel@chu-bordeaux.fr
Pellegrin
I
I
Garrigue
I
I
Caumont
A
A
Schrive
M H
MH
Birac
V
V
Bonot
P
P
Fleury
H
H
D019380:Anti-HIV Agents / Q000494:pharmacology / Q000627:therapeutic use; D024882:Drug Resistance, Viral / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000295:instrumentation; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA / Q000295:instrumentation
D019380:Anti-HIV Agents; D011933:Reagent Kits, Diagnostic; D054303:HIV Reverse Transcriptase; D016333:HIV Protease