Iitiä A, Dahlen P, Nunn M, Mukkala V M, Siitari H
Pharmacia Genetic Engineering, La Jolla, California 92037.
Anal Biochem. 1992 Apr;202(1):76-81. doi: 10.1016/0003-2697(92)90209-p.
Since its discovery, the polymerase chain reaction (PCR) has been used for different purposes in the field of DNA research. We tested the PCR for the diagnosis of HTLV-I/-II infections. PCR was used to amplify 141- and 149-base pair regions from the HTLV-I and HTLV-II virus genomes, respectively. The annealing temperature in the PCR amplification was optimized using 20% polyacrylamide gels and silver staining. Even a slight change (3 degrees C) in the annealing temperature had an effect on the specificity of the reaction. The PCR products were detected with biotin and Eu-labeled oligonucleotide probes in a solution hybridization format. The linearity of the assay was tested with serial dilutions of purified chromosomal DNA containing integrated HTLV-II sequences. The linearity was found to be dependent on the number of cycles used in the PCR amplification. The best linearity, at a target level of a few copies, was achieved using a low number of cycles. The specificity of the assay was tested using HTLV-I and HTLV-II-infected lymphocytes from the cell lines Hut102 and MO480, respectively. No cross reactivity between these analytes was observed.
自聚合酶链反应（PCR）被发现以来，它已在DNA研究领域用于不同目的。我们测试了PCR用于诊断HTLV-I/-II感染。PCR分别用于扩增来自HTLV-I和HTLV-II病毒基因组的141个和149个碱基对区域。使用20%聚丙烯酰胺凝胶和银染对PCR扩增中的退火温度进行了优化。即使退火温度有轻微变化（3摄氏度）也会对反应的特异性产生影响。PCR产物在溶液杂交形式中用生物素和铕标记的寡核苷酸探针进行检测。用含有整合的HTLV-II序列的纯化染色体DNA的系列稀释液测试了该检测方法的线性。发现线性取决于PCR扩增中使用的循环数。在低循环数下，在目标水平为几个拷贝时实现了最佳线性。分别使用来自细胞系Hut102和MO480的HTLV-I和HTLV-II感染的淋巴细胞测试了该检测方法的特异性。未观察到这些分析物之间的交叉反应。
