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疾病发作之前提供额外的一周警告。在前驱阶段较早的时间点开始抗病毒治疗可能会降低患者发生活动性巨细胞病毒病的机会。我们已经完成了检测棕黄色外套细胞中CMV的竞争性定量PCR方法的建立。该检测方法可以在扩增反应管中检测到少至三到五个病毒基因组等价物。该方法的变异系数约为40%，与其他已发表的这类分析方法的描述一致。为了提高检测的精确度，从而对疾病的发生或进展有更好的潜在预测价值，我们开发了一种实时巨细胞病毒聚合酶链式反应检测方法。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。该方法的扩增和检测可在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。对前瞻性研究数据的初步分析表明，实时荧光定量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.