Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay
Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay
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DOI：
10.1128/jcm.01515-06
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发表时间：
2007-05-01
影响因子：
9.4
通讯作者：
Kimura, Hiroshi
中科院分区：
文献类型：
--
作者：
Wada, Kaoru;Kubota, Naomi;Kimura, Hiroshi
我们利用6-羧基荧光素、6-羧基4′、5'-二氯-2′、7'-二甲基荧光素和碳青素5标记探针，建立了多重实时PCR检测方法，同时定量eb病毒（EBV）、巨细胞病毒（CMV）和人类疱疹病毒6 （HHV-6）的DNA。当先前测试和存储的DNA样本被检查时，多重实时PCR分析的结果与单一实时PCR分析的结果一样敏感和特异性。采用多重测定法定量46例移植受者的EBV、CMW和HHV-6 DNA。共采集全血和血浆标本303份。结果显示，EBV全血和血浆病毒DNA检出率分别为23.8%和5.9%，CMW为11.2%和5.3%，HHV-6为12.5%和2.0%。所有形式的病毒DNA在全血中的检测频率都高于血浆。在症状期，所有全血标本中检测到EBV DNA，但未在所有血浆标本中检测到EBV DNA。此外，症状期全血中EBV DNA载量高于无症状期，而两期血浆中EBV DNA载量相似。这些结果表明，全血更适合于移植患者EBV DNA的定量。然而，一个与临床相关的临界值仍有待确定。
We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimetboxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMW, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMW, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.