Development of a Polymerase Chain Reaction Procedure for Quantitative Measuremen

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。除了继续进行实时PCR检测的开发工作外，我们还开始对Organon Teknika NASBA pp67 CMV检测进行评估。这种商业上可用的方法利用逆转录酶和RNA聚合酶在等温反应中扩增CMV RNA。通过靶向病毒mRNA，它应该能够检测复制的病毒，而不会从可能存在的整合病毒DNA拷贝中产生阳性信号。我们收集了几个患者的连续血样集，以评估NASBA pp67 CMV检测结果与先前获得的pp65 CMV抗原血症结果的关系。我们还将使用LightCycler实时聚合酶链式反应来测试这些样本集，以便我们可以将其性能与其他两种方法进行比较。

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. In addition to continuing the developmental work on a real-time PCR assay, we have begun performing an evaluation of the Organon Teknika NASBA pp67 CMV assay. This commercially available assay amplifies CMV RNA in an isothermal reaction using reverse transcriptase and RNA polymerase. By targeting viral mRNA, it should permit detection of replicating virus without the generating positive signal from integrated viral DNA copies that may be present. We have collected sequential blood sample sets from several patients for an evaluation of the NASBA pp67 CMV assay performance against previously obtained pp65 CMV antigenemia results. We also will use the LightCycler real-time PCR to test these sample sets so that we can compare its performance with the other two methods.