Development Of A Polymerase Chain Reaction Procedure For

巨细胞病毒（CMV）疾病是免疫功能低下的巨细胞病毒感染患者中较为常见且严重的并发症。在过去的几年里，很明显，为了区分亚临床病毒脱落和大规模病毒复制（发生在活动性疾病发作前的前驱期），有必要使用序列监测和定量分析。多项研究表明，CMV定量聚合酶链反应（PCR）检测方法比褐皮CMV抗原检测方法更敏感。在某些情况下，这种额外的敏感性可以在巨细胞病毒疾病发作前提供额外的一周警告。在前驱期较早的时间点进行抗病毒治疗可能会减少患者发展为活动性巨细胞病毒疾病的机会。我们已经完成了一种有竞争力的定量PCR检测方法的开发，用于检测褐皮细胞中的巨细胞病毒。该试验可以在扩增反应管中检测到三到五个病毒基因组等价物。该分析的方差系数约为40%，与其他已发表的此类分析的描述一致。为了提高检测精度，从而更好地预测疾病的发生或进展，我们开发了一种实时CMV PCR检测方法。本实验利用频率共振能量转移荧光探针，设计用于Roche LightCycler。扩增和检测可在45至50分钟内完成。我们进行了一项前瞻性研究，利用实时CMV PCR检测骨髓移植患者的全血样本。对前瞻性研究数据的初步分析表明，实时PCR检测对CMV抗原检测的病毒血症事件具有高度敏感性，对CMV抗原阴性标本的阴性预测率大于95%。

Cytomegalovirus (CMV) disease is a relatively frequent, and often serious, complication in immunocompromised, CMV-infected patients. In the past few years, it has become apparent that to differentiate between subclinical viral shedding and large-scale viral replication, occurring during the prodrome before the onset of active disease, it is necessary to use sequential monitoring with a quantitative assay. Several studies have shown that CMV quantitative polymerase chain reaction (PCR) assays are more sensitive than buffy coat CMV antigen detection assays. This extra sensitivity can, in some cases, give an additional week of warning before the onset of CMV disease. Instituting antiviral therapy at an earlier time point in the prodromal stage may decrease the chance of the patient developing active CMV disease. We have completed development of a competitive quantitative PCR assay for the detection of CMV in buffy coat cells. The assay can detect as few as three to five viral genome equivalents in an amplification reaction tube. The coefficient of variance of this assay is about 40 percent, in line with other published descriptions of assays of this type. To have an assay with improved precision and, therefore, better potential predictive value for disease onset or progression, we have developed a real-time CMV PCR assay. This assay utilizes frequency resonance energy transfer fluorescence probes and is designed to run on the Roche LightCycler. Amplification and detection of the assay can be completed within 45 to 50 minutes. We have conducted a prospective study utilizing the real-time CMV PCR assay to test whole blood samples from bone marrow transplant patients. Preliminary analysis of the prospective study data reveals that the real-time PCR assay has a high degree of sensitivity for detecting viremic episodes which are detected by CMV antigen and a negative predictive of greater than 95% for CMV antigen negative specimens.