Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay
Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay
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DOI：
10.1128/jcm.41.8.3840-3845.2003
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发表时间：
2003-08-01
影响因子：
9.4
通讯作者：
Izopet, J
中科院分区：
文献类型：
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作者：
Mengelle, C;Sandres-Sauné, K;Izopet, J
血液中人巨细胞病毒(HCMV)DNA的检测正在成为监测免疫抑制和非免疫抑制患者HCMV感染的标准方法。由于可以使用不同的血室，我们比较了实时荧光聚合酶链式反应检测全血、外周血白细胞和血浆中的人巨细胞病毒DNA。我们检测了286份样本：用Magna Pure仪器(罗氏分子生物化学)自动从WB和PBL中提取HCMV DNA，并从血浆样本中手动提取HCMV DNA。用实时荧光定量光循环聚合酶链式反应检测外周血、外周血和血浆中的人巨细胞病毒DNA。引物和探针均位于UL 83区。白带(88.5%)高于外周血(65.7%)(P&0.0001)或血浆(55.2%)(P<；0.0001)。WB和PBL的阳性结果之间有很好的相关性(r=0.68；P<0.0001)，每200,000个PBL中有3.15log(10)个基因组拷贝，相当于每200,000个白细胞中有50pp65阳性的多形核细胞的阈值，相当于每200个WB中有3.4log(10)个基因组拷贝。通过对不同人群代表性患者的系列样本的分析，表明WB适合于实时荧光定量光周期PCR检测人巨细胞病毒DNA的自动化提取和定量。该系统对免疫抑制和非免疫抑制患者的监测可能非常有用。
The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log(10) genome copies in 200,000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200,000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 mul of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients.