Caballero O L, Menezes C L, Costa M C, Fernandes S C, Anacleto T M, de Oliveira R M, Viotti E A, Távora E R, Vilaça S S, Sabbaga E, de Paula F J, Távora P F, Villa L L, Simpson A J
Ludwig Institute for Cancer Research, Departamento de Bioquímica, Instituto de Química, USP, São Paulo, Brazil.
J Clin Microbiol. 1997 Dec;35(12):3192-7. doi: 10.1128/jcm.35.12.3192-3197.1997.
A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10(6) leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.
本文描述了一种用于检测人巨细胞病毒（HCMV）DNA的多重单步PCR方法。该方法扩增病毒LA和IE基因区域，并在试剂混合和引物退火时采用高温，同时通过聚丙烯酰胺凝胶银染进行产物检测。此检测方法可在含有高达100 ng人DNA的临床样本中检测到1至5个HCMV基因组，其灵敏度与涉及巢式PCR或扩增后杂交的更复杂检测方法相当。除了在需要高灵敏度定性诊断的临床情况下具有重要意义外，该检测方法还适用于监测肾移植受者的HCMV感染。由于其多重检测形式，该检测方法可提供定量信息，即仅扩增一个靶标的样本每10(6)个白细胞中平均含有的病毒基因组比扩增两个靶标的样本少7倍。对肾移植患者每周一次的血液白细胞DNA样本进行检测时，连续三次检测中两个HCMV靶标均被扩增的结果高度提示患者已出现非常高的HCMV载量（灵敏度100%，特异性88%）。因此，我们表明，这种允许进行高灵敏度HCMV诊断的简单PCR检测方法也可用于有效识别有临床显著感染风险的移植受者。
