Epstein-Barr virus quantitation by real-time PCR targeting multiple gene segments - A novel approach to screen for the virus in paraffin-embedded tissue and plasma
Epstein-Barr virus quantitation by real-time PCR targeting multiple gene segments - A novel approach to screen for the virus in paraffin-embedded tissue and plasma
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DOI：
10.1016/s1525-1578(10)60535-1
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发表时间：
2004-11-01
影响因子：
4.1
通讯作者：
Gulley, ML
中科院分区：
文献类型：
--
作者：
Ryan, JL;Fan, HX;Gulley, ML
EB病毒（EBV）感染几乎所有人，然后持续宿主的生命。在一些后来患上癌症的人中，EBV DNA存在于恶性细胞中，并在血浆中以升高的水平循环。在目前的研究中，我们验证了五种新的定量聚合酶链反应（Q-PCR）检测靶向不同但高度保守的EBV基因组片段（BamH 1 W，EBNA 1，LMP 1，LMP 2和BZLF 1）。每种检测方法对每个反应中少至50个拷贝的EBV DNA敏感，并且在至少四个数量级上呈线性。当应用于石蜡包埋的组织与EBV编码的RNA（EBER）原位杂交，BamHI W和EBNA 1检测是最翔实的，而使用整个电池的EBV PCR检测可能有助于确定基因组多态性或缺失。与13例EBER阴性肿瘤相比，在17例EBER阳性肿瘤中发现了更高的病毒载量（平均每100，000个细胞中分别有84，978和22个EBV拷贝）。5个Q-PCR检测也提供了血浆样本中的信息，其中EBV在所有9名淋巴瘤或传染性单核细胞增多症患者中可测量，而EBV在所有9名健康对照中不可检测。研究结果表明，Q-PCR是一种有效的方法来区分疾病相关病毒从偶发病毒在石蜡包埋组织和血浆样品。
Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamHI W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.