Objectives: To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method.
Methods: The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed.
Results: When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05).
Conclusions: The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.
目的: 检验玻璃珠-涡旋振荡改良法提升硅藻DNA提取的效果。方法: 以DNeasy PowerSoil Pro试剂盒为对照，另选取2种不同原理的植物DNA提取试剂盒（新型植物基因组DNA提取试剂盒、Plant DNA Isolation试剂盒）和1种血液DNA提取试剂盒（全血基因组DNA提取试剂盒）对同一水样及同一溺死案件肺组织的硅藻DNA进行提取。设计不同大小玻璃珠质量比和涡旋振荡时间的组合，选择加入玻璃珠振荡的最佳DNA提取条件。将常规组与改良组的提取产物直接进行电泳，经硅藻特异性PCR扩增检测，采用qPCR对提取物进行定量，对各组的Ct值进行统计学分析。结果: 当涡旋振荡频率为3 000 r/min时，DNA提取的最佳组合为涡旋振荡4 min，大小玻璃珠质量比为1∶1。以DNeasy PowerSoil Pro试剂盒的Ct值为参照，10 mL水样的Ct值大于0.5 g组织的Ct值。其他3种试剂盒采用玻璃珠-涡旋振荡改良法后，用于植物DNA提取的2种试剂盒Ct值均下降，且改良前后组织的Ct值差异均有统计学意义（P<0.05）；全血基因组DNA提取试剂盒可以成功提取硅藻DNA，水样提取效果接近DNeasy PowerSoil Pro试剂盒，改良法用于组织样本后，其Ct值差异有统计学意义（P<0.05）；但应用3种试剂盒对水样中硅藻DNA进行提取时，改良前后的Ct值只有新型植物基因组DNA提取试剂盒差异具有统计学意义（P<0.05）。结论: 玻璃珠-涡旋振荡改良法可提高植物及血液DNA提取试剂盒对法医学检材中硅藻DNA的提取效果，特别是对组织样本中硅藻DNA的提取。.
Keywords: DNA extraction; diatom; drowning; forensic pathology; glass bead-vortex oscillation improving method.