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目的:建立一种强直性脊柱炎基因(HLA-B27)的荧光定量PCR(FQ-PCR)方法。方法:扩增强直性脊柱炎患者白细胞中的HLA-B27基因与质粒PGEM-TEasy Vector重组,转化大肠杆菌E.coli DH5α,获得克隆的HLA-B27基因标准模板。用DA7600 PCR仪检测FQ-PCR扩增产物制成标准曲线来定量检测标本中HLA-B27的浓度。结果:FQ-PCR扩增产物呈“S”形动力学曲线;ct(循环阈值)与PCR体系中起始模板拷贝数的对数值之间有较好的线性关系,显示了FQ-PCR定量的准确性。结论:FQ-PCR方法用于定量检测外周静脉血HLA-B27基因表达水平,具有较好的灵敏度、特异性,且操作简便、快速,易于普及,有良好的应用前景。
Objective: To establish an FQ-PCR method for ankylosing spondylitis gene (HLA-B27). Methods: The HLA-B27 gene in leukocyte from patients with ankylosing spondylitis was amplified by recombination with plasmid PGEM-TEasy Vector and transformed into E. coli DH5α. The standard template of HLA-B27 gene was cloned. The DA7600 PCR instrument was used to detect the concentration of HLA-B27 in the sample by quantitatively determining the concentration of HLA-B27 in the standard curve obtained by FQ-PCR amplification. RESULTS: The FQ-PCR products showed a “S” shape kinetic curve. There was a good linear relationship between the ct (cycle threshold) and the logarithm of the initial template copy number in the PCR system, indicating that FQ-PCR Quantitative accuracy. Conclusion: The FQ-PCR method can be used to quantitatively detect the expression of HLA-B27 in peripheral venous blood. It has good sensitivity and specificity and is simple, rapid and easy to popularize. It has good application prospect.