CAJ | 학술논문

目的建立面肩肱型肌营养不良(FSHD)的实时荧光定量PCR(FQ-PCR)检测方法.方法常规酚-氯仿法抽提基囡组DNA,通过EcoR Ⅰ酶切及琼脂糖凝胶电泳,回收38 kb以下DNA作为模板,根据4号染色体上D424序列设计特异的引物和探针,对115例研究对象进行FQ-PCR检测,根据荧光曲线与阳性对照的比较判断结果.结果 16例已知EcoR Ⅰ片段大小的FSHD患者FQ-PCR检测结果为13例阳性,78名健康人检测结果除3例阳性外其余均为阴性,16例经临床诊断的新FSHD患者和5名高危者中分别有15例和3例检测结果为阳性.统计学分析FQ-PCR方法与传统印迹杂交方法(κ=0.765,P=0.002)及临床诊断(κ=0.844,P=0.000)之间的一致性,结果有统计学意义.结论我们创建了以FQ-PCR技术对FSHD进行基因诊断的新方法.该方法能克服印迹杂交方法费时费力、有放射性污染的缺点,且能较好解决由于4q-10q易位及p13E-11探针结合部位缺失造成传统杂交基因诊断方法欠准确的问题,具有较好的临床应用价值.
목적 건립면견굉형기영양불량(FSHD)적실시형광정량PCR(FQ-PCR)검측방법.방법 상규분-록방법추제기닙조DNA,통과EcoR Ⅰ매절급경지당응효전영,회수38 kb이하DNA작위모판,근거4호염색체상D424서렬설계특이적인물화탐침,대115례연구대상진행FQ-PCR검측,근거형광곡선여양성대조적비교판단결과.결과 16례이지EcoR Ⅰ편단대소적FSHD환자FQ-PCR검측결과위13례양성,78명건강인검측결과제3례양성외기여균위음성,16례경림상진단적신FSHD환자화5명고위자중분별유15례화3례검측결과위양성.통계학분석FQ-PCR방법여전통인적잡교방법(κ=0.765,P=0.002)급림상진단(κ=0.844,P=0.000)지간적일치성,결과유통계학의의.결론 아문창건료이FQ-PCR기술대FSHD진행기인진단적신방법.해방법능극복인적잡교방법비시비력、유방사성오염적결점,차능교호해결유우4q-10q역위급p13E-11탐침결합부위결실조성전통잡교기인진단방법흠준학적문제,구유교호적림상응용개치.
Objective To develop a convenient, rapid and specific method using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) for detection of facioscapulohumeral muscular dystrophy(FSHD). Methods Genomic DNA was extracted and digested by restricted endonuclease EcoR Ⅰ , followed by agarose electrophoresis. The DNA (< 38 kb) was retrieved from agarose electrophoretic gels. The primers and probe were designed in D4ZA gene in chromosome 4. One hundred and fifteen subjects were examined by FQ-PCR using the retrieved DNA (<38 kb) as a template and the result was analyzed by fluorescent curve comparing with positive control. Results The results by FQ-PCR showed that 13 cases were positive in 16 FSHD cases whose EcoR Ⅰ fragment sizes were known, 75 cases were negative in 78 cases of normal controls, 15 cases were positive in 16 FSHD cases diagnosed clinically whose EcoR Ⅰ fragment sizes were unknown, and 3 cases were positive in 5 cases of relatives of FSHD patients. Consistency was checked using Kappa index between the 2 gene diagnostic tests for FSHD (FQ-PCR test and the traditional Southern blotting test), and between the 2 diagnostic criterions (gene diagnosis by FQ-PCR and clinical diagnosis). The results were statistically significant (κ = 0. 765, P = 0. 002 ; κ = 0. 844, P = 0. 000). Conclusions A new genetic diagnostic method of FSHD by FQ-PCR was developed, which was more simplified and reliable compared to the time-consuming, radioactive Southern blotting. It could also detect the D4Z4 arrays in cases having deletion of p13E-11 as well as the interchromosomal exchange between 4q35 and 10q26. The new method of FQ-PCR for FSHD may be extended to utilize clinically in future.