Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences.
Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences.
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DOI：
10.1186/1742-4690-7-109
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发表时间：
2010-12-20
期刊：
影响因子：
3.3
通讯作者：
Huber BT
中科院分区：
文献类型：
--
作者：
Oakes B;Tai AK;Cingöz O;Henefield MH;Levine S;Coffin JM;Huber BT
2006年，在一些前列腺肿瘤中发现了一种新型γ逆转录病毒XMRV（异种鼠白血病病毒相关病毒）。最近的一项研究表明，67% 的慢性疲劳综合症 (CFS) 患者可以检测到这种传染性逆转录病毒，但只有极少数健康对照者 (4%) 可以检测到这种传染性逆转录病毒。然而，迄今为止，几个小组已发表文章称，他们无法在其他 CFS 患者群体中识别 XMRV RNA 或 DNA 序列，而另一个小组在 87% 的此类患者中检测到鼠白血病病毒 (MLV) 样序列，但在健康对照中只有 7%。由于 XMRV 和丰富的内源性 MLV 原病毒之间存在高度相似性，因此区分污染性小鼠序列与真正的感染非常重要。使用两种不同的 PCR 检测方法对 112 名 CFS 患者和 36 名健康对照者的外周血 DNA 进行了 XMRV 检测。针对 XMRV pol 序列的 TaqMan qPCR 检测能够在高达 5 μg 的人类基因组 DNA 中检测到来自 2 个 XMRV 感染细胞的病毒 DNA（约 10-12 pg DNA），但在对来自所有测试样本的 100,000 个外周血细胞的 600 ng 基因组 DNA 进行检测时，得出阴性结果。然而，使用针对不同 XMRV 序列的特异性较低的巢式 PCR 检测，其中一些样本获得了阳性结果。 PCR 产物的 DNA 测序揭示了多种病毒相关序列，其中一些与前列腺癌和慢性疲劳综合症患者中发现的序列相同，另一些则与已知的内源性 MLV 关系更密切。然而，所有 XMRV 和/或 MLV DNA 检测呈阳性的样本也对高度丰富的脑池内 A 型颗粒 (IAP) 长末端重复序列呈阳性，并且大多数对鼠线粒体细胞色素氧化酶序列呈阳性。在任何阴性对照样品（包含无 DNA 模板的样品）中均未观察到污染，这些样品均包含在每次测定中。每个小鼠细胞含有超过 100 个拷贝的内源性 MLV DNA。使用高度灵敏的 PCR 技术，即使远少于一个细胞的 DNA 也可以产生可检测的产物。因此，在所有测试样品中通过足够灵敏的检测来监测小鼠 DNA 的污染至关重要。
In 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%). However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true infections. DNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from 2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 μg of human genomic DNA, but yielded negative results in the test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence. DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase sequences. No contamination was observed in any of the negative control samples, containing those with no DNA template, which were included in each assay. Mouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cell's worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.
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影响因子：
3.7
作者：
Erlwein O;Kaye S;McClure MO;Weber J;Wills G;Collier D;Wessely S;Cleare A
通讯作者：
Cleare A
影响因子：
3.3
作者：
Groom HC;Boucherit VC;Makinson K;Randal E;Baptista S;Hagan S;Gow JW;Mattes FM;Breuer J;Kerr JR;Stoye JP;Bishop KN
通讯作者：
Bishop KN
影响因子：
4.5
作者：
Jern P;Stoye JP;Coffin JM
通讯作者：
Coffin JM
影响因子：
56.9
作者：
Lombardi, Vincent C.;Ruscetti, Francis W.;Mikovits, Judy A.
通讯作者：
Mikovits, Judy A.
DOI：
10.1136/bmj.c1018
发表时间：
2010-02-25
期刊：
BMJ (Clinical research ed.)
影响因子：
--
作者：
van Kuppeveld FJ;de Jong AS;Lanke KH;Verhaegh GW;Melchers WJ;Swanink CM;Bleijenberg G;Netea MG;Galama JM;van der Meer JW
通讯作者：
van der Meer JW