Fluorotyping of HLA-DRB by sequence-specific priming and fluorogenic probing
Fluorotyping of HLA-DRB by sequence-specific priming and fluorogenic probing
复制标题
DOI：
10.1034/j.1399-0039.1999.530312.x
复制
发表时间：
1999-03-01
期刊：
影响因子：
--
通讯作者：
Blasczyk, R
中科院分区：
文献类型：
--
作者：
Albis-Camps, M;Blasczyk, R
与我们最近描述的 HLA-A 和 -C 荧光分型策略类似，本研究的目的是开发一种基于序列特异性引物聚合酶链式反应 (PCR-SSP) 的 HLA-DRB 荧光分型方法。应用荧光 5' 核酸酶测定，可以通过废除所有劳动密集型的扩增后步骤来提高样品通量率。此外，还消除了与污染相关的问题。该方法依赖于 Taq-DNA 聚合酶的 5'-3' 核酸外切酶活性，该酶在成功的 PCR 过程中切割目标特异性且单独标记的荧光探针。可以在单次 PCR 中应用针对不同靶标的不同标记探针，从而可以独立检测特定的 HLA 和内部对照产物。用于检测 HLA-DRB 特异性扩增子的探针在其 5' 端用 FAM 作为报告分子进行标记，并在 3' 端用 TAMRA 作为猝灭剂进行标记。探针在第二个外显子内与被所有引物混合物覆盖的保守区域杂交。在扩增的情况下，荧光探针的裂解导致 TAMRA 介导的猝灭效应中断，并产生 FAM 荧光的显着增加。 HLA-DRB 荧光分型信息基于 24 个单独的引物混合物释放的 FAM 荧光。 TET-TAMRA 标记的探针用于指示每个 PCR 反应中内部对照序列的扩增。到目前为止，使用这种荧光分型方法对代表所有血清学定义的 HLA-DRB 特异性的 170 个 PCR 分型临床样本进行了分析。结果与常规琼脂糖凝胶检测结果100%一致。
Similar to our recently described HLA-A and -C fluorotyping strategies, the aim of this study was to develop a sequence-specific primed polymerase chain reaction (PCR-SSP)-based fluorotyping method for HLA-DRB. Applying the fluorogenic 5' nuclease assay, it is possible to increase the sample throughput rate by abolishing all labor-intensive post-amplification steps. Additionally, problems related to contamination are eliminated, The method relies on the 5'-3' exonuclease activity of the Taq-DNA Polymerase which cleaves a target-specific and individually labelled fluorogenic probe during successful PCR. Different labelled probes specific for different targets can be applied in a single PCR, allowing independent detection of the specific HLA and the internal control product. The probe used to detect the HLA-DRB specific amplicons was labeled at its 5' end with FAM as the reporter and further 3' with TAMRA as the quencher, The probe hybridized within the 2nd exon to a conserved region which was covered by all primer mixes. In case of amplification, the cleavage of the fluorogenic probe led to an interruption of the TAMRA-mediated quenching effect and generated a significant increase of the FAM fluorescence. The HLA-DRB fluorotyping information was based on the FAM fluorescence released by 24 individual primer mixes. A TET-TAMRA-labelled probe was used to indicate amplification of the internal control sequence in each PCR reaction. So far, 170 PCR typed clinical samples representing all serologically defined HLA-DRB specificities were analyzed using this fluorotyping method. The results were 100% concordant with those obtained by conventional agarose gel detection.