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Journal of Clinical Microbiology, September 2005, p. 4773-4779, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4773-4779.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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, and Norman L. Letvin*

Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Research East Room 113, 330 Brookline Avenue, Boston, Massachusetts 02215

Received 31 March 2005/ Returned for modification 18 June 2005/ Accepted 25 June 2005

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.

* Corresponding author. Mailing address: Harvard Medical School, Beth Israel Deaconess Medical Center, Division of Viral Pathogenesis, Research East Room 113, 330 Brookline Avenue, Boston, MA 02215. Phone: (617) 667-2766. Fax: (617) 667-8210. E-mail: nletvin{at}bidmc.harvard.edu.

Journal of Clinical Microbiology, September 2005, p. 4773-4779, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4773-4779.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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