Bustin Stephen A
The Royal London Hospital, Centre for Academic Surgery, 4th Floor Alexandra Wing, London, E1 1BB, UK.
Expert Rev Mol Diagn. 2005 Jul;5(4):493-8. doi: 10.1586/14737159.5.4.493.
Real-time fluorescence-based quantitative PCR has become established as the benchmark technology for the quantification of nucleic acids, offering an immense choice of protocols, chemistries and instruments. However, whilst there are comparatively few technical problems associated with DNA-targeted quantitative PCR, this is not the case for real-time reverse transcription PCR assays, and there is considerable uncertainty regarding biological or clinical relevance of many real-time reverse transcription PCR results. A survey of working practices of nearly 100 delegates carried out prior to the Third qPCR Symposium held in London, UK, April 25-26, 2005, reveals some of the reasons underlying the variability of reported real-time reverse transcription PCR results. Specifically, the survey reveals extensive interlaboratory variation in assay design, validation and analysis that, together with other dubious practices, are the likely cause for the publication of variable results. These results emphasize the urgent need for the establishment of best practice guidelines for this technology, particularly in the context of its mounting adaptation as a high-throughput clinical diagnostic assay.
基于实时荧光的定量PCR已成为核酸定量的基准技术，提供了大量的方案、化学方法和仪器选择。然而，虽然与DNA靶向定量PCR相关的技术问题相对较少，但实时逆转录PCR检测并非如此，而且许多实时逆转录PCR结果的生物学或临床相关性存在相当大的不确定性。在2005年4月25日至26日于英国伦敦举行的第三届定量PCR研讨会之前，对近100名与会代表的工作实践进行的一项调查揭示了报告的实时逆转录PCR结果存在差异的一些原因。具体而言，该调查揭示了检测设计、验证和分析方面广泛的实验室间差异，这些差异与其他可疑做法一起，可能是导致结果差异发表的原因。这些结果强调了迫切需要为该技术建立最佳实践指南，特别是在其作为高通量临床诊断检测方法越来越多地被采用的背景下。
