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An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

机译:使用标准的XMRV引物扩增商业RT-PCR试剂盒中的内源性鼠白血病病毒基因组污染物

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摘要

During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.
机译:在初步研究中研究了日本慢性疲劳综合症(CFS)患者血清中异种鼠白血病病毒(MLV)相关病毒(XMRV)感染病毒RNA的存在时,经常在日本预期的产品尺寸上检测到阳性条带使用一步式RT-PCR试剂盒检测XMRV的部分gag区域时,阴性对照样品。我们怀疑试剂盒本身可能已被少量内源性MLV基因组或XMRV污染,并试图在两个独立的实验室中评估试剂盒的质量。我们从日本的Invitrogen,TaKaRa,Promega和QIAGEN购买了四个单步RT-PCR试剂盒。为了扩增XMRV或其他MLV相关病毒的部分gag基因,使用了在XMRV研究中广泛使用的引物组(419F和1154R,以及GAG-I-F和GAG-I-R)。确定了扩增子的核苷酸序列,并将其与来自CFS患者的多型内源性MLV(PmERV),XMRV和内源性MLV相关病毒的保藏序列进行了比较。我们发现,来自Invitrogen的一步式RT-PCR试剂盒的酶混合物被衍生自PmERV的RNA污染。通过RT-PCR扩增的污染物的部分gag区域的核苷酸序列与7号染色体上的PmERV几乎相同(99.4%一致性),与最近发现的CFS患者衍生的MLV样病毒高度相似(96.9%至97.6%) 。我们还确定了污染物的部分env区的核苷酸序列,发现它与PmERV几乎相同(99.6%)。在研究CFS和前列腺癌患者的XMRV感染时,研究人员应在进行PCR和RT-PCR测试之前,谨慎评估试剂盒中是否存在内源性MLV以及XMRV基因组。

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