DEVELOPMENT OF A POLYMERASE CHAIN REACTION PROCEDURE FOR QUANTITATIVE MEASUREMENT

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和严重的并发症。在过去的几年里，为了区分亚临床病毒脱落和在活动性疾病发作前的前驱期发生的大规模病毒复制，有必要利用定量分析的顺序监测。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患者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继续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。每个PCR管的反应混合物中包含标准量的DNA靶序列模拟物，以便检测管对管PCR效率的变化，并在计算由测量的信号强度产生的病毒拷贝数时考虑到这一点。该试验能够在扩增反应中检测到三到五个病毒基因组等价物。在一系列患者样本中，定量PCR方案与p65抗原血症测定的初步比较表明，PCR检测具有更高的灵敏度，并且可以在CMV疾病发病前早期检测到CMV前驱症状。我们试验的方差系数（CV）约为40%，与其他已发表的此类试验描述一致。为了开发一种性能更好的检测方法，从而更好地预测患者疾病发生或进展，我们开发了第二种实时PCR检测方法。这种设计的分析通常有10%或更少的cv。这种改进的实时PCR检测结合了几项刚刚可用的新技术进步。该分析使用了一种新的仪器，LightCycler，它使用气流加热和冷却毛细管，循环时间非常快。仪器还测量荧光强度（阳性信号），从每一个毛细管作为分析运行。我们还指定了一种杂交探针，用于利用最近描述的分子信标配置的CMV定量检测。该系统的初步研究取得了成功。计划用一系列血液样本进行验证研究。

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.