Cytomegalovirus: Quantitative Polymerase Chain Reaction

巨细胞病毒：定量聚合酶链反应

• 批准号: 6542076
• 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/6542076
• 关键词: Herpesviridae disease; blood tests; communicable disease control; communicable disease diagnosis; cytomegalovirus; diagnosis design /evaluation; diagnosis quality /standard; diagnostic tests; disease /disorder onset; disease /disorder proneness /risk; early diagnosis; fluorescence resonance energy transfer; fluorescent dye /probe; human subject; method development; patient care planning; patient oriented research; polymerase chain reaction; time resolved data; virus load

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. A retrospective study was conducted comparing the performance of the real time quantatitive CMV PCR assay with the NASBA pp67 and pp65 antigen detection assays. The real-time CMV PCR assay was found to detect all the significant episodes of CMV viremia identified by the pp65 antigen assay, but also gave a positive signal at least a week earlier in about 75% of the episodes. The pp67 NASBA assay was less sensitive than the other two methods. These results were presented at the American Society for Microbiology meeting in May, 2001. We have initiated a prospective study utilizing the real-time CMV PCR assay to test whole blood samples from bone marrow transplant patients.

巨细胞病毒 (CMV) 疾病是免疫功能低下的 CMV 感染患者中相对常见且通常严重的并发症。在过去几年中，很明显，为了区分亚临床病毒脱落和活动性疾病发作前前驱症状期间发生的大规模病毒复制，有必要使用定量检测进行序贯监测。多项研究表明，CMV 定量聚合酶链反应 (PCR) 检测比血沉棕黄层 CMV 抗原检测检测更灵敏。在某些情况下，这种额外的敏感性可以在 CMV 疾病发作前额外提供一周的警告。在前驱期的较早时间点开始抗病毒治疗可能会降低患者发展为活动性巨细胞病毒疾病的机会。我们已经完成了竞争性定量 PCR 检测的开发，用于检测血沉棕黄层细胞中的 CMV。该测定可以在扩增反应管中检测到少至三到五个病毒基因组当量。该测定的变异系数约为 40%，与其他已发表的此类测定的描述一致。为了提高检测精度，从而对疾病发作或进展具有更好的潜在预测价值，我们开发了实时 CMV PCR 检测。该测定利用频率共振能量转移荧光探针，设计用于在 Roche LightCycler 上运行。检测的扩增和检测可在 45 至 50 分钟内完成。除了继续实时 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。这种市售检测方法使用逆转录酶和 RNA 聚合酶在等温反应中扩增 CMV RNA。进行了一项回顾性研究，比较了实时定量 CMV PCR 测定与 NASBA pp67 和 pp65 抗原检测测定的性能。实时 CMV PCR 检测发现可以检测 pp65 抗原检测所识别的所有 CMV 病毒血症的显着发作，而且在至少一周前约 75% 的发作中也给出了阳性信号。 pp67 NASBA 测定的灵敏度低于其他两种方法。这些结果在 2001 年 5 月的美国微生物学会会议上公布。我们发起了一项前瞻性研究，利用实时 CMV PCR 检测来检测来自骨髓移植患者的全血样本。