面肩肱型肌营养不良实时荧光定量PCR诊断方法的影响因素分析  

Factors influencing diagnosis of facioscapulohumeral muscular dystrophy by real-time fluorescent quantitative PCR

作　　者：苏全喜[1] 李婉仪[2] 张成[3] 熊符[4] 洪铭范[1] 刘爱群[1] 田素娟[5] 

机构地区：[1]广东药学院附属第一医院神经内科,广州510080 [2]东莞市人民医院神经内科,东莞523018 [3]中山大学附属第一医院神经科,广州510080 [4]南方医科大学基础医学院医学遗传学教研室,广州510515 [5]广东药学院生命科学与生物制药学院,广州510006

出　　处：《军医进修学院学报》2011年第7期748-751,共4页Academic Journal of Pla Postgraduate Medical School

基　　金：国家自然科学基金项目(30870851);广东省自然科学基金面上项目(8151008018000002)~~

摘　　要：目的分析探讨面肩肱型肌营养不良(FSHD)实时荧光定量聚合酶链反应(FQ-PCR)诊断方法的影响因素。方法通过改变镁离子终浓度、PCR扩增中退火温度、退火时间或延伸时间、RNase H酶用量和活性等,确定FQ-PCR诊断FSHD的最佳反应条件。结果退火温度55℃、时间20s、镁离子终浓度5.0mM、RNase H酶用量100U、RNase H酶在95℃下灭活8min为最佳反应条件,其中RNase H的活性选择对FSHD诊断的特异性影响较大。结论通过降低RNase H酶的活性可提高诊断的特异性,最终确立了FSHD的FQ-PCR基因诊断最佳反应条件。Objective To analyze the factors influencing diagnosis of facioscapulohumeral muscular dystrophy（FSHD） by real-time fluorescent quantitative polymerase chain reaction（FQ-PCR）. Methods Optimal reaction conditions of FQ-PCR for the diagnosis of FSHD were established by changing the annealing temperature, annealing and elongation time of FQ-PCR, as well as the Mg2^＋ concentration, the amount of RNase H used and its activity. Results The optimal reaction conditions of FQ-PCR for the diagnosis of FSHD were as follows. The Mg2＋ concentration was 5.0mM, the used RNase H was 100U, the PCR annealing time was 20s and the temperature was 55 ℃, the inactivating time of RNase H was 8min and the temperature was 95℃ . Of these factors, the activity of RNase H influenced the specificity of FQ-PCR more significantly for the diagnosis of FSHD. Conclusion The optimal reaction conditions of FQ- PCR for the diagnosis of FSHD can be established by increasing the specificity of FQ-PCR through decreasing the activity of RNase H.

关 键 词：肌营养不良 面肩肱型 实时荧光定量PCR RNASE H酶活性 

分 类 号：R746.2[医药卫生—神经病学与精神病学]