应用SYBR GreenⅠ实时荧光聚合酶链反应检测SpA患者人类白细胞抗原-B27

Detection of HLA-B27 by SYBR GreenⅠbased RT-PCR

目的评价SYBR GreenⅠ实时荧光聚合酶链反应检测人类白细胞抗原-B27。方法对65例SpA患者和100例健康人群,分别以PCR-SSP和SYBR GreenⅠ荧光PCR检测外周血HLA-B27 DNA进行检测,比较两种检测方法。结果丳CR-SSP检测HLA-B27阳性样本呈现135bp和268两个条带,阴性标本仅有268bp一条条带。SYBR GreenⅠRT-PCR检测结果示,阳性标本的熔解曲线呈现HLA-B27的90.37℃和β-globin的86.71℃两个Tm峰,阴性标本仅有β-globin一个Tm峰;侾CR-SSP和SYBR GreenⅠRT-PCR结果符合率为100%;僑pA患者和健康人的HLA-B27阳性率分别为70.76%和6%。结论 SYBR GreenⅠRT-PCR是一种可靠、快速的检测HLA-B27方法。

Objective：To evaluate the methods of SYBR GreenⅠbased real-time PCT which detects the expression of HLA-B27 gene in SpA patients.Methods：DNA samples from 65 SpA patients and 100 healthy persons were detected by SYBR GreenⅠbased real-time fluorescent PCR and sequence specific primer PCR（PCR-SSP）.Results：①In PCR-SSP,136bp fragment and 268bp fragment were both detected in the HLA-B27 positive samples,and only 268bp fragment was found in the HLA-B27 negative sample.In real-time PCR,HLA-B27-positive samples gave two melting curve peak at 90.37℃ for HLA-B27 and 86.71℃ for β-globin,whereas negative samples only had two melting curve peak at 86.71℃ for β-globin.②HLA-B27 genotyping detection by SYBR GreenⅠRT-PCR was completely concordant with the results of PCR-SSP.③The positive rate of HLA-B27 was 70.76%（52/65） and 6%（9/100） in SpA patients and healthy individuals.Conclusion：SYBR GreenⅠreal-time fluorescent PCR is a rapid and reliable method to genotype HLA-B27.