Hamann Lutz, Hamprecht Axel, Gomma Abuzeid, Schumann Ralf R
Institute for Microbiology and Hygiene, Charite Medical Center, Humboldt University, Dorotheenstrasse, Berlin 96 10117, Germany.
J Immunol Methods. 2004 Feb 15;285(2):281-91. doi: 10.1016/j.jim.2003.12.005.
The discovery of the human Toll-like receptors (TLRs) and the recognition of their pivotal role in sensing microbial pathogens during the last 5 years have resulted in a renewed appreciation of innate immunity. Due to their central role in both, triggering innate immunity as well as linking innate and adaptive immunity, genetic variations within the TLR genes, known to be associated with a variety of infectious diseases, are currently of great interest. Several single nucleotide polymorphisms (SNPs) within TLR genes have been described and seem to be associated with susceptibility to inflammatory diseases. However, methods for genotyping SNPs within the TLR genes, e.g. direct sequencing or polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis, are time-consuming. In this work, we report novel real-time PCR methods for genotyping five TLR SNPs within TLR-2, TLR-4 and TLR-9 that have been associated with various diseases using fluorescence labeled hybridization probes and the LightCycler instrument. In addition, we provide protocols employing a standard Taq polymerase in order to reduce substantially the costs for real-time PCR.
在过去5年中，人类Toll样受体（TLR）的发现及其在感知微生物病原体方面关键作用的确认，使人们对固有免疫有了新的认识。由于它们在触发固有免疫以及连接固有免疫和适应性免疫方面都起着核心作用，已知TLR基因内的遗传变异与多种传染病相关，目前备受关注。TLR基因内的几个单核苷酸多态性（SNP）已被描述，似乎与炎症性疾病的易感性有关。然而，TLR基因内SNP的基因分型方法，如直接测序或基于聚合酶链反应（PCR）的限制性片段长度多态性（RFLP）分析，都很耗时。在这项工作中，我们报告了使用荧光标记杂交探针和LightCycler仪器对TLR-2、TLR-4和TLR-9内五个与各种疾病相关的TLR SNP进行基因分型的新型实时PCR方法。此外，我们提供了使用标准Taq聚合酶的方案，以便大幅降低实时PCR的成本。
