Peyerl Fred W, Barouch Dan H, Bazick Heidi S, Manuel Edwin, Letvin Norman L
Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
J Clin Microbiol. 2005 Sep;43(9):4773-9. doi: 10.1128/JCM.43.9.4773-4779.2005.
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.
细胞毒性T淋巴细胞（CTL）对慢病毒施加的免疫压力会选择病毒CTL表位突变。目前用于监测新出现的CTL表位突变的方法依赖于病毒群体或克隆测序这种劳动强度大且耗时的技术。在此，我们描述了一种高通量定量逆转录PCR检测方法的开发，该方法有助于大规模的CTL表位监测。这种方法同时利用了序列特异性分子信标和不依赖序列的双链DNA结合染料SYBR Green。我们表明，该检测方法能检测从感染猿猴免疫缺陷病毒（SIV）的恒河猴的病毒培养上清液和血浆样本中分离出的病毒RNA中免疫显性CTL表位的单核苷酸突变。此外，即使在含有10,000个总拷贝的样本中突变病毒仅占500个突变拷贝时也能检测到。这种用于评估CTL表位突变的实时PCR技术可能被证明是监测感染个体中人类免疫缺陷病毒和SIV基因漂移的有用工具。
