正交试验优化实现毛细管中的快速高效DNA提取

目的利用正交试验优化毛细管DNA提取的实验条件，并对优化后的毛细管DNA提取法进行考察。方法结合液滴与磁珠控制方法，在聚四氟乙烯毛细管中依次完成进样、DNA结合、洗涤以及洗脱等过程，建立毛细管DNA提取法。对照方法采用传统煮沸裂解法。采用正交试验考察血液乙型肝炎病毒（HBVDNA）提取的实验条件，包括结合时间、洗脱时间与洗脱温度等对DNA提取效果的影响。考察毛细管DNA提取法的DNA提取效果与重现性，分析提取的DNA定量（Ct值）与原始样品浓度的相关性。比较毛细管DNA提取法与对照方法提取DNA定量结果及耗时。结果正交试验提示毛细管DNA提取法的最佳实验条件为磁珠DNA结合时间5min，洗脱温度60℃，洗脱时间1min。荧光定量PCR显示，毛细管DNA提取法DNA提取效果较好，回收DNA定量具有良好的重现性，提取的DNA定量与原始样品浓度呈正相关（r=0．9999，P〈0．01）。配对t检验显示，毛细管DNA提取法的回收DNA定量显著高于对照方法（t=-4．263，P〈0．001）。采用优化的实验条件，整个毛细管DNA提取操作在23min内完成，而对照方法耗时45min以上。结论利用正交试验实现了毛细管DNA提取实验参数的优化，建立了一种快速、高效的DNA提取方法。

Rapid and highly efficient DNA extraction in a capillary through orthogonal test optimization

Objective To optimize the experimental conditions for in-capillary DNA extraction through orthogonal test and to evaluate the optimized DNA extraction. Methods By using a protocol that controls droplet with magnetic beads, the in-capillary DNA extraction method was developed which involved sample introduction, DNA binding, magnetic beads rinsing and elution of DNA sequentially processed in a polytetrafluoroethylene （PTFE） capillary. Conventional cleavage by boiling was used as control approach. The orthogonal test was used to study the experimental conditions for DNA extraction of blood hepatitis B virus （HBV）, which included the effects of DNA binding time, elution time and elution temperature on the performance of the in-capillary DNA extraction. Efficiency and reproducibility of the in-capillary DNA extraction assay, as well as the dependence of Ct value of extracted DNA on input DNA concentration were also appraised. The quantitative DNA recovery and time needed for DNA extraction were compared between the capillary-based and conventional methods. Results DNA binding for 5 min and e!ution at 60 C for 1 min was the optimal experimental condition for in-capillary method showed by the orthogonal test. Fluorescent quantitative PCR showed good repeatability of DNA recovery and positive correlation of the DNA production with the amount of HBV DNA input （r=0.9999, P〈0.01）. Paired t-test analysis showed significantly higher DNA recovery using the capillary method compared to the conventional one （t=-4.263, P〈0.001）. Under optimized condition, the entire process of DNA extraction could be completed in 23 min vs 45 min or longer with conventional approach. Conclusion The experimental condition of in-capillary DNA extraction is optimized successfully using orthogonal test, and a rapid and highly efficient DNA extraction mothod is established.