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 unders tanding 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 z idovudine (ZDV), We describe a technique for the detection of the T215Y mut ation using reverse transcription-polymerase chain reaction (RT-PCR) amplif ication of viral sequences and a 5' nuclease assay requiring fluorogenic pr obes. 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 DN A sequencing, and the convenience of combining RT-PCR virus amplification w ith the allelic discrimination assay, without the need for purification of PCR products.