DEVELOPMENT OF A POLYMERASE CHAIN REACTION PROCEDURE FOR QUANTITATIVE MEASUREMENT

巨细胞病毒(CMV)病是免疫功能低下的CMV感染者的一种相对常见且通常严重的并发症。在过去的几年中，为了区分亚临床病毒脱落和活动疾病发作前的前驱症状期间发生的大规模病毒复制，有必要利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患者出现CMV病之前额外提供一周的警告。在前驱阶段的较早时间点进行抗病毒治疗可能会降低患者继续发展为活动性巨细胞病毒病的机会。我们已经建立了检测棕黄色涂层细胞中CMV的竞争性定量聚合酶链式反应方法。在每个聚合酶链式反应管的反应混合物中包括标准量的DNA靶序列的模拟物，从而检测管对管聚合酶链式反应效率的变化，并在根据测量的信号强度计算病毒拷贝数时考虑在内。在扩增反应中，该方法能够检测到少至三到五个病毒基因组的等价物。定量聚合酶链式反应方法与p65抗原血症检测方法在一系列患者样本中的初步比较表明，聚合酶链式反应方法具有更高的灵敏度，并允许在巨细胞病毒病发病之前更早地检测到巨细胞病毒的前驱症状。本方法的变异系数(CV)约为40%，与其他已发表的这类分析方法的描述一致。为了开发一种性能更好的分析方法，从而对患者的疾病发生或进展有更好的潜在预测价值，我们开发了实时PCR分析的第二个版本。此设计的分析通常具有10%或更少的CV。这种改进的实时聚合酶链式反应分析结合了几项刚刚问世的新技术进步。该检测使用了一种名为LightCycler的新仪器，该仪器利用气流来加热和冷却毛细管，以实现非常快的循环时间。该仪器还在分析运行时测量来自每个毛细管的荧光强度(正信号)。我们还指定了一种杂交探针用于CMV定量检测，该检测利用了最近描述的分子信标配置。对该系统的初步研究取得了成功。对一系列血液样本的验证研究正在计划中。

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 detec-tion 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 so that variations in tube-to-tube PCR efficiency are detected and accounted for in 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 quanti-tative 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 pre-dictive value for disease onset or progression in patients, we have developed a second version of a real-time PCR assay. Assays of this design often have CVs of 10 percent or less. This improved real-time PCR assay incorporates several new technical advances that have just become available. The assay utilizes a new instrument, the LightCycler, which uses airflow to heat and cool capillary tubes for very rapid cycling times. The instrument also measures fluorescence intensity (positive signal) from each capillary tube as the assay is running. We have also designated a hybridization probe for use with the quantitative CMV assay that uti-lizes the recently described molecular beacon configuration. Preliminary studies with this system have been successful. Validation studies with a series of blood samples are planned.