【摘要】 目的 建立滤纸干血斑基因组DNA自动化提取方法及流程，并探索其在脊髓性肌萎缩症(SMA)基因筛查中的应用。方法 收集45份干血斑标本，采用打孔钳打孔后提取DNA，使用多重PCR结合荧光探针技术进行SMN1检测，考察最低起始干血斑用量。收集103份4℃不同保存时长的新生儿干血斑和17份EDTA抗凝血制备的干血斑，使用最低起始量提取DNA并检测SMN1，考察运输与保存条件对基因检测结果稳定性的影响。结果 最低1片直径4mm的干血斑所提取的DNA浓度2.54～5.71ng/μL、纯度1.676～1.984，可满足SMN1检测要求。4℃避光保存1～24个月的新生儿干血斑标本，新鲜采集并经冷链运输48～72h的EDTA抗凝血及-80℃超低温冷冻保存1～2年的EDTA抗凝血制备的干血斑标本均可稳定检出SMA基因型。结论 1片直径4mm的干血斑即可满足SMN1检测要求，大大降低了样本采集、保存和运输的难度和复杂程度，有利于开展大规模人群筛查，为现有新生儿筛查体系下增加SMA筛查提供了技术基础。更多还原

【Abstract】 Objective To establish an automatic extraction method of genomic DNA from dried blood spots(DBS), and to explore its application in genetic screening of spinal muscular atrophy(SMA). Methods 45 DBS specimens were collected and punched for genomic DNA extraction. Multiplex PCR combined with fluorescent probe technology was used for SMN1 testing to determine the minimum starting amount of dried blood spot. A total of 103 neonatal DBS specimens stored at 4°C for different months and 17 DBS specimens prepared from EDTA anticoagulants were collected respectively, and the minimum starting amount of these samples were used for genomic DNA extraction and SMN1 testing to investigate the effects of blood transport and storage conditions for SMN1 testing. Results One dried blood spot(4mm in diameter) was proved to be the minimum starting amount for SMN1 testing. The genomic DNA concentrations ranged from 2.54～5.71ng/μL, with a purity of 1.676～1.984. DBS can be stored stably at 4°C for 1～24 months for SMN1 testing. SMA genotype can also be stably detected in DBS specimens prepared from EDTA anticoagulation freshly collected and transported in the cold chain for 48～72 hours or EDTA anticoagulation stored at-80°C for 1 to 2 years.Conclusion The minimum starting amount for SMN1 testing is only one blood spot with a diameter of 4 mm, which greatly reduces the difficulty and complexity of sample collection, storage and transportation. It is very conducive to large-scale population genetic screening of SMA. In particular, it makes it feasible to expand SMA newborn screening under current newborn screening system.更多还原