DEVELOPMENT OF POLYMERASE CHAIN REACTION FOR CYTOMEGALOVIRUS, HERPES SIMPLEX VIRU

在免疫受损的宿主中诊断脑膜炎是困难的，例如那些患有艾滋病或癌症的宿主，因为没有检测最可能的病原体的敏感方法。在这些环境中的生物，如巨细胞病毒(CMV)、单纯疱疹病毒(HSV)和水痘-带状疱疹病毒(VZV)，很难从脑脊液(CSF)中培养出来，并且没有好的抗原检测技术可用。在过去的几年里，越来越清楚的是，用聚合酶链式反应(PCR)方法检测这些病毒病原体可能提供最敏感和最特异的诊断测试。因此，我们已经开发了一套针对CMV、HSV和VZV的内部PCR检测方法，以便将它们作为NIH患者脑脊液的常规服务检测。1997年，脊髓液中巨细胞病毒的聚合酶链式反应方法的建立和临床验证完成，并开始作为常规的分子诊断方法提供。1998年完成了脊髓液中单纯疱疹病毒聚合酶链式反应试验的最终开发和验证，现在该试验也作为一种常规的分子诊断试验提供。对脊髓液的HSV检测将特异性地确定HSV1、HSV2或两者的存在。将继续开发具有与现有CMV和HSV相同性能特征的VZV检测方法。所有这三种检测方法都是为了利用内部模拟物来评估单个PCR管内扩增的效率，并检测PCR抑制，以消除这种假阴性结果的来源。用铕标记的荧光杂交探针进行终点检测，可以检测到三到五个病毒基因组等价物的下限。对巨细胞病毒和单纯疱疹病毒检测的评价显示出极高的特异性。分子诊断技术的灵敏度和可靠性不仅取决于所使用的扩增和检测方法的效率，还取决于所使用的样品制备方法的性能。在过去的一年里，开展了一个大型项目，以调查从人类脊髓液、呼吸样本或血液中提取核酸的六种不同方法的相对效率和可靠性。我们已经确定了一种似乎高度可靠的样品制备方法，并提供了更高的DNA回收效率。这种方法现已被纳入提供的临床分子诊断分析。

Diagnosis of meningitis in immunocompromised hosts, such as those with AIDS or cancer, is difficult because of the unavailability of sensitive methods for detecting the most likely pathogens. Organisms in these settings, such as cytomegalovirus (CMV), Herpes simplex virus (HSV) and Varicella-Zoster virus (VZV), are difficult to grow from cere-brospinal fluid (CSF), and no good antigen detection techniques are available. Over the past few years, it has become increasingly clear that detecting these viral agents by the polymerase chain reaction (PCR) method may provide the most sensitive and specific diagnostic tests. Therefore, we have undertaken development of a battery of in-house PCR assays for CMV, HSV, and VZV so they will be available as routine service tests for CSF from NIH patients. In 1997 development and clinical validation of the PCR assay for CMV in spinal fluid was completed and the test began to be offered as a routine molecular diagnostic assay. During 1998 final development and validation of the PCR assay for HSV in spinal fluid was completed and the test is now also offered as a routine molecular diagnostic assay. The HSV assay on spinal fluid will specifically determine the presence of either HSV1, HSV2, or both viral DNAs. Development of a VZV assay with the same performance characteristics as the existing CMV and HSV assays will continue. All three assays are designed to utilize internal mimics to evaluate the efficiency of amplification within individual PCR tubes and to detect PCR inhibition, in order to eliminate this source of false negative results. Endpoint detection by europium-labeled fluorescence hybridization probes allows a lower limit of detection of three to five viral genome equivalents. Evaluations of the CMV and HSV assays show excel-lent specificity. The sensitivity and reliability of molecular diagnostic techniques depends not only on the efficiencies of the amplification and detection methods utilized, but also on the performance of the sample preparation methods used. In the past year a large-scale project was undertaken to investigate the relative efficiencies and reliabilities of six different methods for preparing nucleic acids from human spinal fluid, respiratory samples or blood. We have identified a sample preparation method that appears to be highly reliable and offers improved efficiency of DNA recovery. This method has now been incorporated into the clinical molecular diagnostic assays offered.