应用不同DNA提取试剂盒对溺死硅藻检验后续分析的影响

Influence of different DNA extraction kits on subsequent diatom analysis in forensic cases of drowning

摘要

目的 研究不同DNA提取试剂盒对水中溺死尸体器官组织中硅藻的分子生物学检测后续分析的影响。 方法 本研究选取经案件侦查、病理学检验，并在电子显微镜下进行硅藻形态学检验确认为溺水死亡的组织及水样，本研究选择了4种不同原理的DNA提取试剂盒，从该死者的肺、肝、脾、肾组织及水样中提取DNA。首先，本研究测量了4 种试剂盒提取产品的核酸含量和测定260 nm和280 nm下吸光度（absorbance，A）值，以评估DNA提取的质量；其次，使用真核18S rDNA V4区域通用引物对提取的产物进行高通量测序，以比较不同DNA提取试剂盒提取的硅藻种类和数量；第三，应用硅藻特异性引物对不同的目的基因进行聚合酶链式反应（polymerase chain reaction，PCR）扩增，以显示不同试剂盒提取不同来源DNA的效果。 结果 水样和组织的硅藻形态学检验共鉴定出19属硅藻。4种DNA提取试剂盒的提取产物A_{260 nm}/A_{280 nm}均低于1.8，PowerSoil试剂盒的相对DNA含量高于其他试剂盒，最低的是BioTeKe试剂盒。高通量测序显示共有49种硅藻，不同试剂盒提取的硅藻DNA的质量和数量不同，不同试剂盒获得的硅藻物种丰富度和优势种群也不同，在4种试剂盒中，PowerSoil试剂盒在水样中提取的硅藻最多，TIANGEN试剂盒从水样中提取的硅藻种类最多，有38种。电泳结果表明，不同试剂盒对提取产物的扩增效果不同，同一试剂盒对不同的样本和不同的目标基因也有不同的扩增效果。 结论 不同原理的试剂盒对后续硅藻分子学水平分析也存在不同效果，在法医学实践中应根据后续分析需求选择最适合的DNA提取试剂盒。

Abstract

Objective To investigate the influence of different DNA extraction kits on the subsequent molecular biological analysis of diatom from the organs and tissues of drowned corpses. Methods This study was conducted using the tissue and water samples confirmed as drowning deaths through case investigation，pathological examination，and morphological examination of diatom under an electron microscope，and four DNA extraction kits with different principles were used to extract DNA from the lung，liver，spleen，and kidney tissues of the deceased and from the water samples from drowning location. Firstly，the products extracted by the four kits were measured in terms of nucleic acid content and A_260nm/A_280nm value to evaluate the quality of DNA extraction； secondly，eukaryotic 18S rDNA V4 universal primers were used to perform high-throughput sequencing of the extracted products，and the results were used to compare the species and numbers of diatoms extracted by different DNA extraction kits； thirdly，PCR amplification was performed for different objective genes using diatom-specific primers to show the effect of DNA from different sources extracted by different kits. Results A total of 19 genera of diatoms were identified based on the morphological analysis of water and tissue samples. The OD260/OD280 value was lower than 1.8 for the products extracted by all four DNA kits； the PowerSoil kit showed a higher relative content of DNA than the other kits，while BioTeKe kit showed the lowest relative content of DNA. A total of 49 diatom species were obtained by high-throughput sequencing，and there were differences in the quality and quantity of diatom DNA extracted by different kits and the abundance and dominant species of diatom obtained by different kits. Among the four kits，PowerSoil kit obtained the highest relative abundance of diatom species in water samples，and TIANGEN kit obtained 38 species of diatom from water samples，which was significantly higher than the number of species obtained by the other kits. The results of electrophoresis showed that different kits had different amplification effects on the extracted products，and the same kit also had different amplification effects on different samples and different objective genes. Conclusion The kits with different principles of DNA extraction have different effects on subsequent molecular biological analysis of diatom，and in forensic practice，the most suitable DNA extraction kit should be selected according to the needs of subsequent analysis.

关键词

DNA提取 / 溺水 / 硅藻 / 高通量测序

Key words

DNA extraction / drowning / diatom / high-throughput sequencing

基金资助

国家自然科学基金资助项目(82060341;81560304)

海南省院士创新平台科研资助项目(YSPTZX202134)

海南医学院研究生创新科研课题资助项目(Qhys2023-444)