Development of a Polymerase Chain Reaction Procedure for Quantitative Measurement

巨细胞病毒(CMV)病是一种相对 免疫功能低下的常见和通常严重的并发症 巨细胞病毒感染患者。在过去的几年里，这一点变得显而易见 为了区分亚临床病毒脱落和 在前驱症状期间发生大规模病毒复制 活动性疾病的发作，有必要利用序贯 用定量的方法进行监测。多项研究表明 CMV定量聚合酶链式反应(PCR)检测 比黄褐色外套CMV抗原检测更敏感。这 在某些情况下，额外的敏感度可能会给你带来额外的一周 在巨细胞病毒病患者发病前发出警告。制定 在先兆阶段的较早时间点进行抗病毒治疗可能 降低患者继续发展为活动性的机会 CMV病。我们已经完成了一种具有竞争力的 定量聚合酶链式反应检测棕黄色大衣中的CMV 细胞。DNA靶序列的标准模拟量是 包括在每个聚合酶链式反应管的反应混合物中 解释了管对管聚合酶链式反应效率的差异 根据测量信号计算病毒拷贝数 力量。这种化验方法可以检测到3到5个。 扩增反应中的病毒基因组等价物。初步 定量聚合酶链式反应与p65抗原血症的比较 一系列患者样本的测定表明， 聚合酶链式反应方法具有更高的敏感性，可以更早地检测到 巨细胞病毒病发病前的前驱症状。这个 本方法的变异系数(CV)约为40%，符合 与其他已发表的这种类型的分析方法的描述。 一种性能更好的检测方法，因此更好 对疾病发病或进展的潜在预测价值 患者们，我们已经研究了OReal-Timeo PCR版本的我们的 化验。此设计的分析通常具有10%或更少的CV。 利用我们的OUR技术建立一种实时定量PCR检测方法 现有有效的引物和探针序列是完整的。

Cytomegalovirus (CMV) disease is a relatively frequent and often serious complication in immunocompromised CMV-infected patients. In the last few years it has become apparent that in order 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 utilize 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 in a patient. Instituting antiviral therapy at an earlier time point in the prodromal stage may decrease the chance that the patient will go on to develop active CMV disease.We have completed development of a competitive quantitative PCR assay for the detection of CMV in buffy coat cells. A standard amount of mimic of the DNA target sequence is included in the reaction mixture of each PCR tube to detect and account for variations in tube-to-tube PCR efficiency in the calculations of viral copy number made from the measured signal strength. The assay is capable of detecting as few as three to five viral genome equivalents in an amplification reaction. Preliminary comparisons of the quantitative PCR protocol with p65 antigenemia determinations in a series of patient samples demonstrates that the PCR assay has greater sensitivity and permits an earlier detection of the CMV prodrome before the onset of CMV disease. The coefficient of variance (CV) of our assay is about 40 percent, in line with other published descriptions of assays of this type.To develop an assay with improved performance, and, therefore, better potential predictive value for disease onset or progression in patients, we have worked on a Oreal-timeO PCR version of our assay. Assays of this design often have CVs of 10 percent or less. Development of one version of a real-time PCR assay using our existing validated primers and probe sequences is complete.