Development Of A Polymerase Chain Reaction Procedure For

巨细胞病毒(CMV)病是免疫功能低下的巨细胞病毒感染患者的一种相对常见且往往严重的并发症。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，在活动性疾病发作之前的前驱症状期间，有必要使用序贯监测和定量检测。多项研究表明，CMV定量聚合酶链式反应(PCR)检测比灰衣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病发病前额外提供一周的警告，并允许在CMV病的前驱阶段的较早时间点进行抗病毒治疗。我们建立了一种实时定量的巨细胞病毒聚合酶链式反应方法。该分析使用频率共振能量转移荧光探针，设计用于罗氏LightCycler上运行。用这种方法可以在45到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式反应检测骨髓移植患者的全血样本。2003财年期间，对前瞻性研究数据的分析表明，实时聚合酶链式反应检测CMV抗原检测的病毒发作具有高度的敏感性，对CMV抗原阴性标本的阴性预测值超过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.