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

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 activityResults 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 FSHDConclusion 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.