Tong Yupin, Pang Xiaoli L, Mabilangan Curtis, Preiksaitis Jutta K
Division of Infectious Diseases, Department of Medicine, and.
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, and.
J Infect Dis. 2017 Apr 1;215(7):1094-1101. doi: 10.1093/infdis/jix069.
Whether cytomegalovirus (CMV) DNA exists in plasma as virion-associated or free DNA is uncertain.
An assay combining DNase I digestion and CMV quantitative polymerase chain reaction (DNase-CMV-qPCR) was developed to differentiate free naked DNA from virion DNA. One hundred three frozen and 10 fresh CMV DNA-positive plasma samples from solid-organ transplant recipients (SOTRs) were tested. Three sets of paired qPCR (P-qPCR) assays with amplicons of variable length were used to study CMV DNA fragmentation in 20 SOTR plasma samples, viral stocks (Towne, Merlin, AD169) and the first World Health Organization (WHO) international standard (IS) for CMV DNA.
In all plasma samples, 98.8%-100% of CMV DNA was free DNA; this was the only form in 93 of 103 (90.3%) frozen and all 10 fresh samples tested using DNase-CMV-qPCR. Low levels of virion CMV DNA were found in 10 of 103 (9.7%) samples with higher total DNA load. Cytomegalovirus DNA results were highly reproducible for 3 CMV virus stocks and WHO IS (P > .80), tested by three sets of paired q-PCR. However, for the 20 SOTR plasma samples, the smaller amplicon assay result was 2.6-fold, 3.4-fold, and 6.5-fold higher than the longer amplicion result (P < .001).
Cytomegalovirus DNA in SOTR plasma is almost exclusively free DNA, highly fragmented, and not virion associated.
巨细胞病毒（CMV）DNA在血浆中是以病毒体相关形式还是游离DNA形式存在尚不确定。
开发了一种结合DNA酶I消化和CMV定量聚合酶链反应（DNase-CMV-qPCR）的检测方法，以区分游离裸露DNA和病毒体DNA。对103份来自实体器官移植受者（SOTR）的冷冻和10份新鲜CMV DNA阳性血浆样本进行了检测。使用三组扩增片段长度可变的配对qPCR（P-qPCR）检测方法，研究了20份SOTR血浆样本、病毒储备液（Towne、Merlin、AD169）以及第一份世界卫生组织（WHO）CMV DNA国际标准品（IS）中的CMV DNA片段化情况。
在所有血浆样本中，98.8%-100%的CMV DNA为游离DNA；在103份冷冻样本中的93份（90.3%）以及使用DNase-CMV-qPCR检测的所有10份新鲜样本中，这是唯一的形式。在103份样本中的10份（9.7%）总DNA载量较高的样本中发现了低水平的病毒体CMV DNA。通过三组配对q-PCR检测，3种CMV病毒储备液和WHO IS的CMV DNA结果具有高度可重复性（P>.80）。然而，对于20份SOTR血浆样本，较小扩增片段检测结果比长扩增片段检测结果分别高2.6倍、3.4倍和6.5倍（P<.001）。
SOTR血浆中的巨细胞病毒DNA几乎完全是游离DNA，高度片段化，且与病毒体无关。
