Use of molecular beacons for rapid, real-time, quantitative monitoring of cytotoxic T-lymphocyte epitope mutations in simian immunodeficiency virus.
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.
Peyerl
Fred W
FW
Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Barouch
Dan H
DH
Bazick
Heidi S
HS
Manuel
Edwin
E
Letvin
Norman L
NL
D000818:Animals; D052160:Benzothiazoles; D002460:Cell Line; D003470:Culture Media; D003959:Diamines; D018984:Epitopes, T-Lymphocyte / Q000235:genetics*; D015683:Gene Products, gag / Q000235:genetics; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics; D008253:Macaca mulatta; D015335:Molecular Probes; D009154:Mutation; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D013997:Time Factors
D052160:Benzothiazoles; D003470:Culture Media; D003959:Diamines; D018984:Epitopes, T-Lymphocyte; D015683:Gene Products, gag; D016056:Immunodominant Epitopes; D015335:Molecular Probes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I