Development of a Polymerase Chain Reaction Procedure for Quantitative Measurement

用于定量测量的聚合酶链式反应程序的开发

• 批准号: 6103699
• 负责人: Steven h FISCHER
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6103699
• 关键词: blood tests; clinical research; cytomegalovirus; human subject; method development; microorganism culture; polymerase chain reaction; virus DNA; virus antigen

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.

巨细胞病毒（CMV）疾病是一种相对较轻的疾病 免疫功能低下患者常见且经常严重的并发症 CMV感染者。在过去的几年里， 为了区分亚临床病毒脱落和 大规模的病毒复制发生在前驱症状之前 在活动性疾病发作时，有必要使用序贯 用定量分析监测。几项研究表明 CMV定量聚合酶链反应（PCR）测定是 比血沉棕黄层CMV抗原检测测定更敏感。这 在某些情况下，额外的敏感性可以提供额外的一周时间， 在患者发生CMV疾病之前发出警告。提起 在前驱期的较早时间点进行抗病毒治疗可 减少患者继续发展为活跃的 CMV疾病。我们已经完成了一个有竞争力的发展， 血沉棕黄层中CMV的定量PCR检测 细胞标准量的DNA靶序列模拟物是 包括在每个PCR管的反应混合物中以进行检测， 解释了管对管PCR效率的变化， 由测量的信号计算病毒拷贝数 实力该检测方法能够检测到三到五个 扩增反应中的病毒基因组等同物。初步 p65抗原血症定量PCR方法的比较 在一系列患者样品中的测定表明， PCR检测具有更高的灵敏度，可以更早地检测出 CMV疾病发病前的CMV前驱症状。的 变异系数（CV）约为40%，符合 与其他已发表的这种类型的测定的描述。 具有改进的性能的测定，并且因此更好地 疾病发作或进展的潜在预测价值 患者，我们已经研究了我们的Oreal-timeO PCR版本， 比色法这种设计的测定通常具有10%或更小的CV。 使用我们的方法开发一种版本的实时PCR测定法， 现有的验证引物和探针序列是完整的。