Cavé H, Acquaviva C, Bièche I, Brault D, de Fraipont F, Fina F, Loric S, Maisonneuve L, Namour F, Tuffery S
Laboratoire de biochimie génétique, Fédération de génétique, Hôpital Robert Debré (AP-HP), 48, boulevard Sérurier, 75019 Paris.
Ann Biol Clin (Paris). 2003 Nov-Dec;61(6):635-44.
Application fields of RT-PCR (reverse transcription-polymerase chain reaction) in clinical diagnosis comprises the assessment of viral load for RNA viruses and the analysis of gene transcription products. RT-PCR is also helpful when large genes have to be sequenced. Developments of quantitative approaches using real-time PCR recently led to a major widening of RT-PCR applications in clinical diagnosis. However, RT reaction is delicate due to its lack of reproducibility and to RNA lability and frequent contamination by DNA. In some cases additional difficulties come from the need to obtain a specific amplification in the presence of homologous sequences which might be present in higher amounts than the sequence of interest. These caveats have to be taken into account, when designing the RT protocol, and when choosing PCR primers and internal and/or external references. This review is aimed at helping the experimental setup of a RT-PCR based assay according to the objectives.
逆转录聚合酶链反应（RT-PCR）在临床诊断中的应用领域包括评估RNA病毒的病毒载量以及分析基因转录产物。当需要对大基因进行测序时，RT-PCR也很有帮助。最近，使用实时PCR的定量方法的发展极大地拓宽了RT-PCR在临床诊断中的应用。然而，由于RT反应缺乏可重复性、RNA不稳定以及频繁受到DNA污染，该反应较为棘手。在某些情况下，额外的困难来自于需要在存在比目标序列含量更高的同源序列的情况下获得特异性扩增。在设计RT方案以及选择PCR引物和内部和/或外部参照时，必须考虑到这些注意事项。本综述旨在根据目标帮助基于RT-PCR的检测方法进行实验设置。
