Development Of A Polymerase Chain Reaction Procedure For

巨细胞病毒（CMV）疾病是一种相对常见的，往往是严重的，在免疫功能低下，CMV感染的患者并发症。在过去的几年中，很明显，要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症状期间，有必要使用定量检测进行连续监测。几项研究表明，CMV定量聚合酶链反应（PCR）检测比血沉棕黄层CMV抗原检测更敏感。在某些情况下，这种额外的敏感性可以在CMV疾病发作前提供额外一周的警告，并允许在CMV疾病前驱期的较早时间点进行抗病毒治疗。我们已经开发了一种定量实时CMV PCR检测。该检测采用频率共振能量转移荧光探针，设计在Roche LightCycler上运行。用该测定法可以在45至50分钟内完成信号的扩增和检测。我们进行了一项前瞻性研究，利用实时巨细胞病毒聚合酶链反应（CMV PCR）检测骨髓移植患者的全血样本。在2003财年期间，前瞻性研究数据的分析显示，实时PCR检测试剂盒对于检测CMV抗原检测的病毒血症发作具有高度灵敏度，并且对于CMV抗原阴性标本的阴性预测值大于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 and allow institution of antiviral therapy at an earlier time point in the prodromal stage of CMV disease. We have developed a quantitative 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 signal with this 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. During FY 2003 analysis of the prospective study data has revealed 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.