Quantitation of human cytomegalovirus in recipients of solid organ transplants by real-time quantitative PCR and pp65 antigenemia
Quantitation of human cytomegalovirus in recipients of solid organ transplants by real-time quantitative PCR and pp65 antigenemia
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DOI：
10.1002/jmv.10277
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发表时间：
2003-02-01
影响因子：
12.7
通讯作者：
Izopet, J
中科院分区：
文献类型：
--
作者：
Mengelle, C;Pasquier, C;Izopet, J
人巨细胞病毒（HCMV）感染和抗HCMV治疗通常通过测量pp 65抗原血症来监测。这种方法耗时，劳动密集，需要熟练的操作人员。我们比较了使用实时光循环仪（TM）定量PCR（QPCR）和pp 65抗原检测从实体器官移植受者收集的系列样品获得的结果。我们收集了14例实体器官移植受者的198份血液样本，用LightCyclerTM PCR检测pp 65抗原。从白细胞中提取HCMV DNA，用位于UL 83区的引物和探针进行检测。使用由含有靶序列的质粒制备的标准曲线计算HCMV DNA的量。pp 65阳性细胞数与DNA拷贝数呈良好的相关性（r = 0.57，P < 0.0001）。50个阳性多形核白细胞/200，000个细胞的临床阈值相当于通过Light Cycler（TM）PCR检测的每毛细管两个log（10）基因组拷贝。HCMV DNA检测前pp 65抗原在3例患者中，平均时间为10天，而这两个测试同时为8例患者呈阳性。在抗病毒治疗期间，pp 65抗原数据和DNA拷贝数均随时间降低，尽管在pp 65抗原测定变为阴性后28.2天QPCR为阳性。实时光循环T定量PCR检测是一种快速、省力的技术。这种分子方法可能有助于监测实体器官移植受者的感染和抗病毒治疗。
Human cytomegalovirus (HCMV) infections and anti-HCMV treatment are usually monitored by measuring pp65 antigenemia. This method is time-consuming, labour-intensive and requires skilled operators. We have compared results obtained using real-time Light Cycler(TM) quantitative PCR (QPCR) and the pp65 antigen assay on serial samples collected from recipients of solid organ transplants. We collected 198 blood samples from 14 solid organ transplant recipients and assayed them for pp65 antigen and with Light Cycler TM PCR. HCMV DNA was extracted from leukocytes and measured using primers and probe locate in the UL83 region. The quantity of HCMV DNA was calculated using a standard curve prepared from a plasmid containing the target sequence. There was a good correlation between the number of pp65-positive cells and the DNA copy number (r = 0.57, P < 0.0001). A clinical threshold of 50 positive polymorphonuclear leukocytes/ 200,000 cells was equivalent to two log(10) genome copies percapillary by Light Cycler(TM) PCR. HCMV DNA was detected before pp65 antigen in three patients at a mean time of 10 days, whereas the two tests were positive simultaneously for eight patients. Both the pp65 antigen data and DNA copy number decreased over time during antiviral treatment, although the QPCR was positive 28.2 days after the pp65 antigen assay a become negative. The real-time Light Cycler T quantitative PCR assay is a rapid and labour-saving technique. This molecular method could be useful for monitoring infections and antiviral treatment in recipients of solid organ transplants.