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  • Title: Use of molecular beacons for rapid, real-time, quantitative monitoring of cytotoxic T-lymphocyte epitope mutations in simian immunodeficiency virus.
    Author: Peyerl FW, Barouch DH, Bazick HS, Manuel E, Letvin NL.
    Journal: J Clin Microbiol; 2005 Sep; 43(9):4773-9. PubMed ID: 16145140.
    Abstract:
    Immune pressure on lentiviruses exerted by cytotoxic T lymphocytes (CTL) selects for virus CTL epitope mutations. Currently employed methods for monitoring emerging CTL epitope mutations rely on the labor-intensive and time-consuming techniques of virus population or clonal sequencing. Here we describe the development of a high-throughput quantitative reverse transcription-PCR assay that facilitates large-scale CTL epitope monitoring. This approach utilizes both sequence-specific molecular beacons and the sequence-independent double-stranded DNA binding dye Sybr Green. We show that this assay detects single-nucleotide mutations in an immunodominant CTL epitope in viral RNA isolated from both viral culture supernatants and plasma samples from simian immunodeficiency virus (SIV)-infected rhesus monkeys. Furthermore, mutant viruses can be detected even when they represent as few as 500 mutant copies in a sample containing 10,000 total copies. This real-time PCR technique for evaluating CTL epitope mutations may prove to be a useful tool for monitoring the genetic drift of human immunodeficiency virus and SIV in infected individuals.
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