HLA-B27基因荧光定量方法的确立与多种方法比较研究  

Establishment of fluorescence quantitative polymerase-chain-reaction method to quantify HLA-B27 gene:a comparative study of multiple methods

作　　者：戎奇吉[1] 孙讳浩 徐冉行[3] 刘东海[3] 

机构地区：[1]慈溪市人民医院检验科,浙江慈溪315300 [2]中国科学院上海生命科学研究院湖州营养与健产业创新中心,浙江湖州313000 [3]宁波市第六医院检验科,浙江宁波315010

出　　处：《中华医院感染学杂志》2015年第16期3601-3603,3612,共4页Chinese Journal of Nosocomiology

基　　金：宁波市自然科学基金资助项目(2010A610064)

摘　　要：目的在原研究的基础上,重新合成Taqman探针,建立一种新的强直性脊柱炎基因HLA-B27荧光定量PCR(FQ-PCR)方法,为临床治疗提供参考依据。方法 2012年1-11月收集的医院强直性脊柱炎282例,优化各种PCR条件,并与多种方法比较,检测强直性脊柱炎及相关的各类疾病,同期选择100名健康体检人群作为阴性对照,采用SYBR法和Taqman特异性探针荧光定量检测法。结果 FQ-PCR生成动力学有关的PCR扩增曲线由荧光信号强度周期数,HLA-B27的标准曲线是循环阈值之间线性关系(ct)和起始拷贝数;高度相关(0.999)揭示FQ-PCR的可靠性;确立的Taqman法有良好的线性和精度并与流式细胞和SYBR法比较,具有良好的相关性(P<0.05)。结论新确立的荧光定量PCR(Taqman)比其他方法更可靠准确,并有很好的应用价值。OBJECTIVE To establish a novel fluorescence quantitative polymerase‐chain‐reaction （FQ‐PCR） method for routine quantification of ankylosing spondylitis gene （HLA‐B27） based on the primary basis of resynthesis of Taqman probe so as to provide guidance for clinical treatment .METHODS From Jan 2012 to Nov 2012 ,a total of 282 patients with ankylosing spondylitis were enrolled in the study .The PCR conditions were optimized and com‐pared with multiple methods ,and the ankylosing spondylitis and related diseases were detected ;100 healthy peo‐ple who received the physical examination during the same period were chosen as the negative controls ,and the SYBR method and Taqman specific probe fluorescence quantitative detection method were employed .RESULTS The FQ‐PCR generated“S”kinetics curve of PCR amplification was constructed by relating the fluorescence signal intensity （△Rn） to the cycle number ;the standard curve of HLA‐B27 was constructed by the linear relationship between the cycle threshold （ct） and the log of starting copy number .The high correlation （0 .999） revealed the reliability of the FQ‐PCR .The established Taqman method possessed favorable linear and accuracy and showed good relationship with the flow cytometry and SYBR method （P〈0 .05） .CONCLUSION The novel FQ‐PCR（Taq‐man）is more reliable and accurate than other methods ,and it is worthy to be promoted .

关 键 词：HLA-B27基因 荧光定量 聚合酶链反应 流式细胞 

分 类 号：R446[医药卫生—诊断学]