Rapid detection of the HIV type 1 reverse transcriptase codon T215Y by reverse transcription-polymerase chain reaction with fluorogenic probes.
Early detection of viral mutations, particularly those mutations associated with cross-resistance to antiretroviral drugs, is critical both for understanding the mechanism of drug resistance and for the clinical management of patients infected with HIV-1. One of the frequently observed mutations in the HIV-1 reverse transcriptase (RT)-coding region is ACC --> TAC at codon 215, resulting in a change of wild-type threonine (T) to tyrosine (Y); this mutation has been associated with decreased phenotypic susceptibility to zidovudine (ZDV). We describe a technique for the detection of the T215Y mutation using reverse transcription-polymerase chain reaction (RT-PCR) amplification of viral sequences and a 5' nuclease assay requiring fluorogenic probes. In addition to detecting the presence of the ACC --> TAC mutation at codon 215, this assay provides an increased ability to detect low levels of mutant species in a mixed population, relative to conventional sequencing. Further advantages of this technique include the rapid and high-throughput nature of the assay, the accuracy of the assay relative to conventional DNA sequencing, and the convenience of combining RT-PCR virus amplification with the allelic discrimination assay, without the need for purification of PCR products.
Ross
L
L
Department of Virology, Glaxo Wellcome, Research Triangle Park, North Carolina 27709, USA. llr8156@glaxowellcome.com
Vavro
C
C
Zimmermann
A
A
St Clair
M
M
D019380:Anti-HIV Agents / Q000627:therapeutic use; D002648:Child; D002675:Child, Preschool; D003062:Codon; D004252:DNA Mutational Analysis / Q000379:methods; D016049:Didanosine / Q000627:therapeutic use; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D005456:Fluorescent Dyes; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D017354:Point Mutation; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015215:Zidovudine / Q000627:therapeutic use
D019380:Anti-HIV Agents; D003062:Codon; D005456:Fluorescent Dyes; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D016049:Didanosine