High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR-SSOP-Luminex method in the Japanese population
High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR-SSOP-Luminex method in the Japanese population
复制标题
DOI：
10.1007/s00251-005-0048-3
复制
发表时间：
2005-11-01
期刊：
影响因子：
3.2
通讯作者：
Inoko, H
中科院分区：
文献类型：
--
作者：
Itoh, Y;Mizuki, N;Inoko, H
我们开发了一种新的高通量、高分辨率基因分型方法，用于检测人类白细胞抗原（HLA）-A、-B、-C和-DRB 1位点的等位基因，方法是将聚合酶链反应（PCR）和序列特异性寡核苷酸探针（SSOP）方案与Luminex 100 xMAP流式细胞术双激光系统相结合，对附着在彩色编码微珠上的荧光标记寡核苷酸进行定量。为了检测日本人群中频率超过0.1%的HLA等位基因，我们针对HLA-A基因座创建了48个寡核苷酸探针，针对HLA-B基因座创建了61个寡核苷酸探针，针对HLA-C基因座创建了34个寡核苷酸探针，针对HLA-DRB 1基因座创建了51个寡核苷酸探针。使用从加州洛杉矶国际HLA DNA交换大学获得的150份多国对照样本，将其亚克隆至质粒载体后，通过与核苷酸测序方法进行比较，确定PCR-SSOP-Luminex方法的准确度。此外，我们对从1，018名日本志愿者中收集的DNA样本进行了用于HLA等位基因分型的PCR-SSOP-Luminex方法。总体而言，使用150个对照样本，基因分型方法显示HLA-A的准确度为85.91%，HLA-B的准确度为85.03%，HLA-C的准确度为97.32%，HLA-DRB 1的准确度为90.67%，在1，018个日本样本中，HLA-A和-C的准确度为100%，HLA-B的准确度为99.90%，HLA-DRB 1的准确度为99.95%。PCR-SSOP-Luminex方法提供了一种简单、准确和快速的方法，可在日本人群中对HLA等位基因进行多重基因分型，达到四位数或更高的分辨率水平。当由一名打字员处理时，从DNA提取到在HLA-A、HLA-B、HLA-C和HLA-DRB 1位点上定义HLA四位数等位基因仅需约5小时。
We have developed a new high-throughput, high-resolution genotyping method for the detection of alleles at the human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 loci by combining polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes (SSOPs) protocols with the Luminex 100 xMAP flow cytometry dual-laser system to quantitate fluorescently labeled oligonucleotides attached to color-coded microbeads. In order to detect the HLA alleles with a frequency of more than 0.1% in the Japanese population, we created 48 oligonucleotide probes for the HLA-A locus, 61 for HLA-B, 34 for HLA-C, and 51 for HLA-DRB1. The accuracy of the PCR-SSOP-Luminex method was determined by comparing it to the nucleotide sequencing method after subcloning into the plasmid vector using 150 multinational control samples obtained from the International HLA DNA Exchange University of California Los Angeles. In addition, we performed the PCR-SSOP-Luminex method for HLA allele typing on DNA samples collected from 1,018 Japanese volunteers. Overall, the genotyping method exhibited an accuracy of 85.91% for HLA-A, 85.03% for HLA-B, 97.32% for HLA-C, and 90.67% for HLA-DRB1 using 150 control samples, and 100% for HLA-A and -C, 99.90% for HLA-B, and 99.95% for HLA-DRB1 in 1,018 Japanese samples. The PCR-SSOP-Luminex method provides a simple, accurate, and rapid approach toward multiplex genotyping of HLA alleles to the four-digit or higher level of resolution in the Japanese population. It takes only approximately 5 h from DNA extraction to the definition of HLA four-digit alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci for 96 samples when handled by a single typist.