Simple detection of point mutations associated with HIV-1 drug resistance.
A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS--PCR) --- a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant--wild-type population. Gene sequencing was carried out simultaneously for comparison. MS--PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS--PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS--PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS--PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest.
Frater
A J
AJ
Jefferiss Research Trust Laboratories, Department of GUM and Communicable Diseases, Division of Medicine, Imperial College School of Medicine at St. Mary's Hospital, London W2 1PG, UK. j.frater@ic.ac.uk
Chaput
C C
CC
Beddows
S
S
Weber
J N
JN
McClure
M O
MO
D000349:Africa; D019380:Anti-HIV Agents / Q000494:pharmacology*; D023241:Antiretroviral Therapy, Highly Active; D004352:Drug Resistance, Microbial / Q000235:genetics*; D018432:Drug Resistance, Multiple; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D019380:Anti-HIV Agents