Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests
Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests
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通过巢式双聚合酶链反应法检测受感染细胞系和献血者外周单核细胞中的人 T 细胞白血病病毒 I 型 (HTLV-I) 原病毒：与 HTLV-I 抗体测试的比较
作者：
C. Matsumoto;S. Mitsunaga;T. Oguchi;Y. Mitomi;T. Shimada;A. Ichikawa;J. Watanabe;K. Nishioka
应用巢式双重聚合酶链式反应(PCR)技术，对培养的人T细胞白血病病毒I型(HTLV-I)前病毒DNA进行检测。该程序包括第一次扩增和第二次扩增，第一次扩增的产物和第一次引物的内侧的引物。使用这种方法，我们证明了检测单模板DNA是可能的。聚合酶链式反应扩增产物经聚丙烯酰胺凝胶电泳法检测，显示3条模板DNA超量的条带，2条适量的条带，1条限量的条带。根据套式双重聚合酶链式反应的结果，粗略估计PBMC中前病毒的数量。以MT-2和Molt-4混合细胞为靶细胞，采用颗粒凝集试验(PA)和间接免疫荧光试验(IF)检测抗HTLV-I抗体，并与Px区套式双重PCR检测结果进行比较。在101份PA阴性的标本中，无论是IF试验还是PCR试验都没有阳性结果。在检测出抗体阳性的155份标本中，有57份同时进行了聚合酶链式反应和间接免疫荧光检测。免疫荧光和聚合酶链式反应检测结果完全一致。因此，IF法最适合于确认目前在大多数诊断实验室和血液中心使用的PA检测。
Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.
DOI：
10.1073/pnas.86.1.232
发表时间：
1989-01-01
影响因子：
11.1
作者：
SHEFFIELD, VC;COX, DR;MYERS, RM
通讯作者：
MYERS, RM