应用巢式 PCR 监测人类巨细胞病毒感染：血浆和白细胞中阳性率的比较及与定量 PCR 的比较。
Monitoring human cytomegalovirus infection with nested PCR: comparison of positive rates in plasma and leukocytes and with quantitative PCR.
机构信息
Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China.
出版信息
Virol J. 2010 Apr 15;7:73. doi: 10.1186/1743-422X-7-73.
BACKGROUND
Human cytomegalovirus (HCMV) infection poses a significant health threat to immunocompromised individuals. Here we performed this study to set up a highly sensitive nested PCR method applicable for detecting HCMV infection in high-risk individuals. In this work, 106 blood specimens from 66 patients with potential HCMV infection were obtained. Total DNA was extracted separately from plasma and peripheral blood leukocytes (PBL) of each sample. HCMV DNA was detected in parallel by nested PCR and quantitative real-time PCR (qRT-PCR), and the results were compared.
RESULTS
Serial dilution test revealed that the detection limit of nested PCR was 180 copies/ml. The nested PCR showed a higher positive rate than qRT-PCR (34.9% vs. 12.3%, p < 0.001). The positive rate of nested PCR based on PBL DNA was significantly higher than that based on plasma DNA (34.9% vs. 18.9%, p = 0.002). Of the 14 patients with serial samples, 11 were positive for HCMV DNA in PBL while only 7 were positive in plasma. Moreover, for each patient, nested PCR using PBL DNA also detected more positive samples than that using plasma DNA.
CONCLUSION
Combined use of nested PCR with PBL DNA is highly sensitive in defining HCMV infection. This assay is particularly useful in the case of quantification not essential.
背景
人巨细胞病毒（HCMV）感染对免疫功能低下的个体构成重大健康威胁。在这里，我们进行了这项研究，以建立一种高度敏感的巢式 PCR 方法，适用于检测高危个体的 HCMV 感染。在这项工作中，我们从 66 名疑似 HCMV 感染的患者中获得了 106 份血液标本。分别从每份样本的血浆和外周血白细胞（PBL）中提取总 DNA。通过巢式 PCR 和实时定量 PCR（qRT-PCR）并行检测 HCMV DNA，并比较结果。
结果
系列稀释试验表明，巢式 PCR 的检测限为 180 拷贝/ml。巢式 PCR 的阳性率高于 qRT-PCR（34.9%比 12.3%，p<0.001）。基于 PBL DNA 的巢式 PCR 的阳性率明显高于基于血浆 DNA 的阳性率（34.9%比 18.9%，p=0.002）。在 14 例具有连续样本的患者中，11 例 PBL 中有 HCMV DNA 阳性，而仅 7 例血浆中阳性。此外，对于每个患者，使用 PBL DNA 的巢式 PCR 也比使用血浆 DNA 检测到更多的阳性样本。
结论
结合使用 PBL DNA 的巢式 PCR 高度敏感地定义 HCMV 感染。在不需要定量的情况下，该检测方法特别有用。
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