Use of molecular beacons for rapid, real-time, quantitative monitoring of cytotoxic T-lymphocyte epitope mutations in simian immunodeficiency virus

Fred W. Peyerl, Dan H. Barouch, Heidi S. Bazick, Edwin Manuel, Norman L. Letvin

Research output: Contribution to journalArticle

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 (STV)-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.

Original languageEnglish (US)
Pages (from-to)4773-4779
Number of pages7
JournalJournal of clinical microbiology
Volume43
Issue number9
DOIs
StatePublished - Sep 1 2005

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ASJC Scopus subject areas

  • Microbiology (medical)

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