围绕FFPE样本特性的DNA纯化纳米磁珠优化

Optimization of DNA purification nano beads specifically for FFPE samples

目的 探讨围绕福尔马林固定石蜡包埋(formalin fixed paraffin embedded, FFPE)样本特性获取更高质量/得率的纳米磁珠核酸提取方案,改进分子病理技术。方法 合成4大类15个小类的备选磁珠,以FFPE样本为中心,筛选高质量/得率磁珠。模拟常规组织、粗针穿刺(肝脏)、纤维支气管镜样本(肺),装管相同张数连片。采用筛选的最佳磁珠与市场出售的常见磁珠试剂提取核酸,横向对比纯化总量、片段大小等质量参数。应用PCR和Sanger验证核酸的下游应用。结果 以FFPE样本DNA为中心筛选自制纳米磁珠,获得最佳性能纳米磁珠总回收率为58.5%±1.58%,5种市售商品化磁珠和3种国产磁珠总回收率为18.68%~40.71%。相同组织量(连片)提取的DNA总量,在模拟常规组织、粗针穿刺和纤维支气管镜样本核酸得率比市售试剂盒提高39.49%~181.72%(P<0.05)。模拟纤维支气管镜样本1张(4μm)总量可达100 ng以上、5张总量可达400 ng以上。结论 以FFPE样本DNA为中心筛选的DNA纯化纳米磁珠,与商品化磁珠相比有较大提升,为临床分子病理检测的质量保证、自动化检测和项目拓展提供空间。

Purpose Focusing on the characteristics of formalin fixed paraffin embedded(FFPE)samples,explored nanomagnetic bead nucleic acid extraction solutions with higher quality/yield and continued to improve molecular pathology technology.Methods Alternative magnetic beads were synthesised in four major categories and 15 sub-categories and we screened to obtain high-quality/yield magnetic beads centred on FFPE samples.Simulated conventional tissues,simulated coarse needle punctures(liver),and simulated fiberoptic bronchoscopy samples(lungs)were sectioned with the same number of serial slices in tubes.The nucleic acids of slices were extracted using the best magnetic beads screened in this study and common commercially available kits,and then perform comparison and purification quality parameters such as total amount and fragment size.The downstream applications of nucleic acids were validated by PCR and Sanger sequencing.Results Screening all homemade nanomagnetic beads centered on the DNA of FFPE samples,the total recoveries of the best performance nanomagnetic beads were obtained to be 58.5%±1.58%,and the total recoveries of five commercially available commercial magnetic beads and three domestic kit magnetic beads ranged from 18.68%to 40.71%.The total amount of DNA(ng)extracted from the same amount of tissue(serial slices),the nucleic acid yield of this study in simulated conventional tissues,simulated coarse needle punctures,and simulated fiberoptic bronchoscopy samples were increased by 39.49%-181.72%compared with those of the commercially available kits(P<0.05).The total amount of extracted nucleic acid from simulated fiberoptic bronchoscopy tissue sections can be more than 100 ng for 1 slice(4μm)and more than 400 ng for 5 slices.Conclusion The DNA purification nanomagnetic beads screened with DNA from FFPE samples have a significant enhancement comparing to the existing commercial bead protocols,and provide space for quality assurance,automated testing,and program expansion for clinical molecular pathology testing.