Deyde Varough M, Gubareva Larisa V
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Mail Stop G-16, 1600 Clifton Road, Atlanta, GA 30333, USA.
Expert Rev Mol Diagn. 2009 Jul;9(5):493-509. doi: 10.1586/erm.09.21.
Pyrosequencing is a high-throughput non-gel-based DNA sequencing method that was introduced in the late 1990s. It employs a DNA sequencing-by-synthesis approach based on real-time measurement of pyrophosphate released from incorporation of dNTPs. A cascade of enzymatic reactions proportionally converts the pyrophosphate to a light signal recorded in a form of peaks, known as pyrograms. Routinely, a 45-60-nucleotide sequence is obtained per reaction. Recent improvements introduced in the assay chemistry have extended the read to approximately 100 nucleotides. Since its advent, pyrosequencing has been applied in the fields of microbiology, molecular biology and pharmacogenomics. The pyrosequencing approach was first applied to analysis of influenza genome in 2005, when it played a critical role in the timely detection of an unprecedented rise in resistance to the adamantane class of anti-influenza drugs. More recently, pyrosequencing was successfully applied for monitoring the emergence and spread of influenza A (H1N1) virus resistance to oseltamivir, a newer anti-influenza drug. The present report summarizes known applications of the pyrosequencing approach for influenza genome analysis with an emphasis on drug-resistance detection.
焦磷酸测序是一种在20世纪90年代末引入的高通量非凝胶DNA测序方法。它采用基于合成的DNA测序方法，实时测量dNTP掺入时释放的焦磷酸。一系列酶促反应将焦磷酸按比例转化为以峰的形式记录的光信号，即所谓的焦磷酸测序图。通常，每个反应可获得45-60个核苷酸的序列。最近检测化学方面的改进已将读长扩展到约100个核苷酸。自问世以来，焦磷酸测序已应用于微生物学、分子生物学和药物基因组学领域。2005年，焦磷酸测序方法首次应用于流感基因组分析，当时它在及时检测对金刚烷类抗流感药物前所未有的耐药性上升方面发挥了关键作用。最近，焦磷酸测序成功应用于监测甲型H1N1流感病毒对新型抗流感药物奥司他韦的耐药性出现和传播情况。本报告总结了焦磷酸测序方法在流感基因组分析中的已知应用，重点是耐药性检测。
