Müller Jens
Institute for Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany.
Methods Mol Biol. 2010;630:319-35. doi: 10.1007/978-1-60761-629-0_20.
Real-time RT-PCR has become the method of choice for automated detection of viral RNA target sequences in the clinical laboratory. Besides commercially available certified test systems, a variety of so-called in-house methods have been described in the literature. Generally, appropriate validation and continuous quality control are mandatory if these in-house-developed assays are used in clinical diagnostics. In this chapter, an in-house HIV-1 real-time RT-PCR assay for blood donor screening is described. The procedure includes the pooling of plasma samples, viral RNA isolation, and subsequent detection of amplification in real-time one-step RT-PCR. The validation considers the specificity, the sensitivity on HIV-1 genomic variants, and the robustness of the assay.
实时逆转录聚合酶链反应（Real-time RT-PCR）已成为临床实验室中自动检测病毒RNA靶序列的首选方法。除了市售的经过认证的检测系统外，文献中还描述了多种所谓的内部方法。一般来说，如果将这些内部开发的检测方法用于临床诊断，适当的验证和持续质量控制是必不可少的。在本章中，描述了一种用于献血者筛查的内部HIV-1实时逆转录聚合酶链反应检测方法。该程序包括血浆样本的合并、病毒RNA的分离以及随后在实时一步逆转录聚合酶链反应中对扩增的检测。验证考虑了该检测方法的特异性、对HIV-1基因组变异体的敏感性以及稳健性。
