Ambinder R F, Lambe B C, Mann R B, Hayward S D, Zehnbauer B A, Burns W S, Charache P
Johns Hopkins Oncology Center, Baltimore, MD 21231.
Mol Cell Probes. 1990 Oct;4(5):397-407. doi: 10.1016/0890-8508(90)90030-4.
We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.
我们设计了合成寡核苷酸引物和杂交探针，用于聚合酶链反应（PCR）扩增和检测爱泼斯坦-巴尔病毒（EBV）核酸序列的杂交。引物序列选自爱泼斯坦-巴尔病毒核抗原-1（EBNA-1）的编码区。使用这些寡核苷酸进行PCR扩增和杂交，实验对象包括标准实验室细胞系，如非洲伯基特淋巴瘤和传染性单核细胞增多症衍生的细胞系，以及最近从骨髓移植受者的临床EBV分离株建立的细胞系。所有检测的EBV细胞系均为阳性。未感染的细胞系、人胎盘DNA或其他病毒未检测到假阳性。检测方法的灵敏度使得在1微克胎盘DNA的背景下能够始终检测到4份EBV基因组拷贝。在两名传染性单核细胞增多症患者和一名病毒相关性噬血细胞综合征患者的外周血单个核细胞DNA提取物中检测到EBV。18名无已知持续性EBV相关疾病且接受门诊手术的EBV血清阳性患者中，有3名患者的外周血单个核细胞DNA提取物中也检测到EBV。在两名移植后淋巴瘤患者和两名患有原发性中枢神经系统淋巴瘤的艾滋病患者的淋巴瘤组织DNA提取物中检测到EBV。在12例无免疫功能低下病史患者的B细胞淋巴瘤标本中未检测到EBV。先前经Southern印迹法显示为EBV阳性的福尔马林固定石蜡包埋霍奇金组织的DNA提取物，经PCR检测也显示为EBV阳性。因此，使用所述的寡核苷酸，PCR可用于检测一系列临床分离株中的EBV。这些寡核苷酸对所有测试的EBV菌株具有广泛的特异性，这可能是EBNA-1 DNA结合域高度保守序列的作用。
