Department of Clinical Laboratory, the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, PR China.
Biosens Bioelectron. 2013 Aug 15;46:80-3. doi: 10.1016/j.bios.2013.02.023. Epub 2013 Feb 24.
Detection of HLA-B27 gene is clinically important due to its strong association with diseases, such as ankylosing spondylitis. Nucleic acid-based biosensors represent a promising clinical tool for gene diagnosis. Surface plasmon resonance (SPR) can be used to monitor DNA-DNA hybridization in real-time and without any prior labeling step. Here, a self-built HLA-B27 positive PCR products spectral SPR imaging system was used for multichannel direct-detection of a specific sequence of the HLA-B27 gene. Thiol-labeled single-stranded oligonucleotide probes, which were proved to be superior to amine-labeled probes in immobilization, were immobilized onto the surfaces of the gold spots on the sensor chip to target the specific sequence in the HLA-B27 gene in blood. SPR measurements were performed with different concentrations of synthetic target DNA sequence. The calibration curve of synthetic target sequence showed a good linear relationship between the concentration of the synthetic target sequence and the shift of the SPR wavelength from 10 nM to 500 nM with a detection limit of 5 nM. The HLA-B27 gene was isolated from human whole blood and amplified using polymerase chain reaction (PCR). PCR products were measured using the SPR imaging system. HLA-B27 positive PCR products showed significant SPR wavelength shift, while synthetic non-complementary sequence and negative PCR products showed no significant SPR wavelength shift. The method is high-specific, high-throughput and label-free.
由于与疾病（如强直性脊柱炎）的强关联，HLA-B27 基因的检测具有重要的临床意义。基于核酸的生物传感器是基因诊断的一种很有前途的临床工具。表面等离子体共振（SPR）可用于实时监测 DNA-DNA 杂交，而无需任何预先标记步骤。在这里，我们使用自建的 HLA-B27 阳性 PCR 产物谱 SPR 成像系统，对 HLA-B27 基因的特定序列进行多通道直接检测。巯基标记的单链寡核苷酸探针在固定化方面优于胺标记的探针，被固定在传感器芯片上的金点表面，以针对血液中的 HLA-B27 基因的特定序列。对不同浓度的合成靶 DNA 序列进行了 SPR 测量。合成靶序列的校准曲线显示，在 10 nM 至 500 nM 的浓度范围内，合成靶序列的浓度与 SPR 波长的移动之间存在良好的线性关系，检测限为 5 nM。从人全血中分离 HLA-B27 基因，并使用聚合酶链反应（PCR）进行扩增。使用 SPR 成像系统对 PCR 产物进行测量。HLA-B27 阳性 PCR 产物显示出明显的 SPR 波长移动，而合成的非互补序列和阴性 PCR 产物则没有明显的 SPR 波长移动。该方法具有高特异性、高通量和无标记的特点。
