Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR
Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR
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DOI：
10.1016/j.jviromet.2004.08.019
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发表时间：
2005-01-01
影响因子：
3.1
通讯作者：
Nair, VK
中科院分区：
文献类型：
--
作者：
Baigent, SJ;Petherbridge, LJ;Nair, VK
开发、优化和验证了实时 PCR 方法，能够以每百万宿主细胞的拷贝数来定量马立克氏病病毒基因组。双重 PCR 在一次反应中测量病毒 meq 基因和宿主卵转铁蛋白基因，从而能够校正添加的样品 DNA 量的差异。病毒基因组的细菌人工染色体（BAC）克隆和带有鸡卵转铁蛋白基因片段的质粒（pGEM-T-ovo）分别用于定量病毒和宿主基因组。这种灵敏且可重复的测定最初是使用鸡淋巴细胞 DNA 建立的，然后通过在反应中加入牛血清白蛋白来适应羽尖 DNA，以克服黑色素的抑制。主要优点是：（1）确定病毒基因组绝对拷贝数，从而能够在样本之间进行有意义的比较； (2) 每百万细胞拷贝数的表达，允许与噬菌斑测定直接相关； (3)使用BAC克隆的病毒全基因组作为标准，有可能使任何病毒基因都可以用作PCR靶标。这是羽尖 MDV 基因组定量的第一份报告，该测定的应用可以显着加深我们对马立克氏病的发病机制、传播、诊断、遗传抗性和疫苗控制的理解。 (C) 2004 Elsevier B.V. 保留所有权利。
A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T-ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease. (C) 2004 Elsevier B.V. All rights reserved.