Development of a Polymerase Chain Reaction Procedure for Quantitative Measuremen

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。除了继续进行实时PCR检测的开发工作外，我们已经开始对Organon Teknika NASBA pp67 CMV检测进行评估。这种市售的分析扩增CMV RNA在等温反应中使用逆转录酶和RNA聚合酶。通过靶向病毒mRNA，它可以在不产生可能存在的整合病毒DNA副本的阳性信号的情况下检测复制的病毒。我们收集了几位患者的连续血液样本集，以评估NASBA pp67 CMV检测与先前获得的pp65 CMV抗原血症结果的性能。我们还将使用LightCycler实时PCR来测试这些样品集，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.