It was reported earlier that a few patients suffering from non-Hodgkin's lymphoma had low amounts of DNA from the so-called fifth human exogenous retrovirus, HRV-5. A sensitive and rational method for large-scale screening for HRV-5 DNA was therefore developed. It is a single-tube nested quantitative PCR (stnQPCR), which uses two functionally isolated primer pairs and one probe target distinct from related endogenous retroviral sequences, yet encompassing known HRV-5 variation, allowing optimal use of sequence conservation. DNA from lymphoma, myeloma, and follicular dendritic cell lines was tested for HRV-5 positivity, as was DNA from whole blood of blood donors, non-Hodgkin's lymphoma and systemic lupus erythematosus patients, as well as DNA from lymph node biopsies of rheumatoid arthritis patients with lymphoma. One blood donor, one systemic lupus erythematosus patient, two previously known positive non-Hodgkin's lymphoma patients, and one rheumatoid arthritis lymphoma patient, came out positive. They had 24, 2, 148, 480 and 30 proviral copies per microg of DNA from PBMC or lymphoma tissue, respectively. During the completion of this work it was reported that HRV-5 is a rabbit endogenous retrovirus (RERV-H), and that HRV-5 positivity was due to presence of rabbit DNA. DNA from six RERV-H/HRV-5 positive samples was therefore retested. Three also contained rabbit mitochondrial DNA. A search for HRV-5 antibodies using synthetic peptides was negative in sera from three RERV-H/HRV-5 positive individuals, as well as in 144 other sera, according with a noninfectious origin of the RERV-H/HRV-5 DNA in human samples. A search for possible sources of rabbit DNA contamination was negative. Methods for prevention of PCR contamination were strictly adhered to. Three samples from RERV-H/HRV-5 positive individuals positive at the Uppsala laboratory were retested at one or two other laboratories, and all three were positive. Two other samples, which were positive in the Riga laboratory, were tested also in London and also found positive. One non-Hodgkin's lymphoma patient was RERV-H/HRV-5 positive in four consecutive samples, showing that positivity was a property of that patient. It is concluded that the stnQPCR developed to detect and quantify minute amounts of RERV-H/HRV-5 DNA is a principle which can be applied widely and HRV-5 is a RERV-H. Its presence in a few human blood samples could not be explained.

译文

:早先有报道称,少数患有非霍奇金淋巴瘤的患者的所谓第五人类外源性逆转录病毒HRV-5 DNA含量低。因此,开发了一种用于大规模筛选HRV-5 DNA的灵敏而合理的方法。它是一种单管嵌套式定量PCR(stnQPCR),使用两个功能分离的引物对和一个与相关内源逆转录病毒序列不同的探针靶标,但包含已知的HRV-5变异,可以最佳地利用序列保守性。测试了来自淋巴瘤,骨髓瘤和滤泡树突状细胞系的DNA的HRV-5阳性,以及来自献血者,非霍奇金淋巴瘤和系统性红斑狼疮患者的全血的DNA,以及来自类风湿性关节炎的淋巴结活检的DNA淋巴瘤患者。一名献血者,一名系统性红斑狼疮患者,两名先前已知的阳性非霍奇金淋巴瘤患者和一名类风湿关节炎淋巴瘤患者呈阳性。每微克来自PBMC或淋巴瘤组织的DNA分别具有24、2、148、480和30个原病毒拷贝。在完成这项工作的过程中,据报道HRV-5是兔内源性逆转录病毒(RERV-H),并且HRV-5阳性是由于兔DNA的存在。因此,对来自六个RERV-H / HRV-5阳性样品的DNA进行了重新测试。三个还包含兔子线粒体DNA。使用合成肽搜索的HRV-5抗体在来自三个RERV-H / HRV-5阳性个体的血清中以及在144个其他血清中均呈阴性,根据RERV-H / HRV-5 DNA的非感染源。人类样本。寻找兔子DNA污染的可能来源是阴性的。严格遵守防止PCR污染的方法。从Uppsala实验室呈阳性的RERV-H / HRV-5阳性个体的三份样品在另外一两个实验室进行了重新测试,所有三份均为阳性。在里加实验室中呈阳性的其他两个样品也在伦敦进行了测试,并且也呈阳性。一名非霍奇金淋巴瘤患者在四个连续样本中均为RERV-H / HRV-5阳性,表明阳性是该患者的财产。结论是开发用于检测和定量微量RERV-H / HRV-5 DNA的stnQPCR是可以广泛应用的原理,HRV-5是RERV-H。无法解释它在一些人类血液样本中的存在。

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