应用SYBR Green Ⅰ实时荧光PCR法对中国汉族人群HLA-B27进行快速基因分型

目的：建立一种快速、灵敏的实时荧光聚合酶链反应（PCR）对中国汉族人群的人白细胞抗原B27（HLA-B27）进行基因分型。方法：采用SYBR GreenⅠ荧光PCR法对120例已定型的标本、230例健康志愿者、54例类风湿因子（RF）阳性患者和36例抗核抗体（ANA）阳性患者的HLA-B27 DNA进行实时检测，并采用熔解曲线对HLA-B27进行基因分型。结果：①熔解曲线分析显示，伊珠蛋白PCR产物的Tm平均值为87．82℃，HLA-B27 PCR产物的Tm平均值为90．54℃，HLA-B27阴性标本仅在87．82℃处存在β-珠蛋白的熔解曲线峰，而阳性标本则于87．82℃和90．54℃处出现两个熔解曲线峰，且△Tm约为2．72℃；②对120个已定型的标本，SYBR Green Ⅰ实时荧光PCR法与传统的PCR—SSP（序列特异性引物）结果完全吻合；③健康志愿者、RF阳性患者和ANA阳性患者HLA-B27 DNA阳性率分别为2．6%（6／230）、3．7％（2／54）和5．6％（2／36）。结论：SYBR Green Ⅰ实时荧光PCR法是一种快速、灵敏的HLA-B27基因分型方法，具有传统的PCR所不可比拟的优点，可取代传统的PCR法用于中国汉族人群的HLA-B27的基因分型，辅助强直性脊柱炎等脊柱关节病的诊断。

Rapid genotyping of HLA-B27 in chinese han population by SYBR Green Ⅰ real time-fluorescent PCR

Objective：To develop a real-time fluorescent PCR genotype HLA-B27 in Chinese Han population rapidly and sensitively. Method： HLA-B27 DNA were detected by SYBR Green Ⅰ real time fluorescent PCR and genotyped with melting curve in genotyped 120 individuals,230 healthy volunteers, 54 RF positive patients and 36 ANA positive patients. Result： 1.The melting curve analysis showed that the average Tins of β-globin PCIR product and HLA-B27 PCR product were 87.82℃ and 90.54℃ respectively. HLA-B27-negative samples gave one melting curve peak at 87.82℃ for β-globin, whereas positive samples had two melting curve peaks , one represented β-globin at 87.82℃ ,the other represented HLA-B27 at 90.54℃ and △Tm was about 3℃. 2.For genotyped 120 sampled, genotypes detected by SYBR Green Ⅰ real-time fluorescent PCR were completely concordant with the results obtained by conventional PCR SSP. 3.The positive rate of HLA B27 was 2. 6%（6/230）,3.7%（2/54）and 5.6 %（2/36）in healthy volunteers, RF-positive patients and ANA-positive patients respectively. Conclusion： SYBR Green Ⅰ real-time fluorescent PCR is a rapid and sensitive method which genotyped HLA-B27 and incomparably superior to conventional PCR-SSP, which make it applicable for substituting conventional PCR-SSP and genotyping HLA-B27 in Chinese Han population and aiding diagnosing spondyloarthropathy, e. g. , ankylosing spondylitis and acute anterior uveitis.