Pozo F, Tenorio A
Servicio de Microbiología Diagnóstica, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
J Virol Methods. 1999 Apr;79(1):9-19. doi: 10.1016/s0166-0934(98)00164-5.
A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection and typing of all human lymphotropic herpesviruses described to date, including Ebstein Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6, variants A and B (HHV6-A, HHV6-B), human herpesvirus 7 (HHV7) and human herpesvirus 8 (HHV8). Oligonucleotide primers were designed to amplify a highly conserved region within the DNA polymerase gene. Each reaction component and thermal cycling parameters were thoroughly standardized to achieve optimal specificity and sensitivity for the PCR assay, which was estimated at about 10-100 molecules for each virus. An internal control, consisting of 100 molecules of a cloned fragment of the porcine pseudorabies herpesvirus (PrV) genome, was included to detect false negative results. To assess suitability and clinical application of the multiplex PCR method, a total of 35 well-characterized specimens, including Kaposi's sarcoma skin lesions, serum, cerebrospinal fluid, saliva and urine samples, were tested. Results obtained suggest this technique could be applied as a sole diagnostic tool in several clinical settings in which herpesviral infection is suspected and differential diagnosis required, avoiding the need to test specimens by separate PCR methods.
开发了一种多重巢式聚合酶链反应（PCR）方法，用于同时检测和分型迄今为止描述的所有人类嗜淋巴细胞性疱疹病毒，包括爱泼斯坦-巴尔病毒（EBV）、巨细胞病毒（CMV）、人类疱疹病毒6型A和B变体（HHV6-A、HHV6-B）、人类疱疹病毒7型（HHV7）和人类疱疹病毒8型（HHV8）。设计了寡核苷酸引物，以扩增DNA聚合酶基因内的一个高度保守区域。对每个反应成分和热循环参数进行了全面标准化，以实现PCR检测的最佳特异性和灵敏度，估计每种病毒的灵敏度约为10-100个分子。加入了一个内部对照，由100个猪伪狂犬病疱疹病毒（PrV）基因组克隆片段分子组成，以检测假阴性结果。为了评估多重PCR方法的适用性和临床应用，共检测了35个特征明确的标本，包括卡波西肉瘤皮肤病变、血清、脑脊液、唾液和尿液样本。获得的结果表明，该技术可作为一种单独的诊断工具，应用于怀疑疱疹病毒感染并需要进行鉴别诊断的多种临床环境中，避免了通过单独的PCR方法检测标本的需要。
