法医学组织病理切片DNA的实时荧光定量分析

Quantitative analysis of DNA by real-time PCR for forensic pathological section

目的:运用TaqMan探针实时荧光定量扩增法,对法医学病理切片进行DNA定量分析,探讨切片组织类型、保存时间对DNA基因组的质量影响。方法:取14例尸检7个年龄段的新鲜组织样本,制作脑、心、肝、肾、脾、肺、肠等7类组织病理切片。以2个7阶拉丁方实验设计,将切片随机分组保存7 d、14 d、30 d、90 d、180 d、360 d、720 d。用硅珠法提取切片DNA,实时荧光PCR进行DNA定量。结果:同一类型组织的不同保存时间的切片DNA含量存在差异(P<0.01),保存1月以内的切片DNA含量下降不显著,半年以上的样本DNA含量下降明显;同一保存时间的不同组织类型之间的DNA含量存在差异(P<0.01),肺、肝、肾三种组织切片DNA含量相对较高;身源年龄组之间差异不显著(P>0.6)。结论:法医学病理组织切片DNA含量较低,为低拷贝模板,组织切片保存时间、组织类型是影响DNA质量的重要因素。

Objective：To investigate the effects of preserving period and tissue types of pathological sections on the quality of DNA genome for forensic science using real-time quantitative PCR detection with TaqMan probe-based gene analysis.Methods：The fresh tissue samples were obtained from 14 cases of autopsy by 7 death ages for preparing pathological sections of the brain,heart,liver,lung,spleen,kidney and ileum tissue,and randomly maintained these sections for 7,14,30,90,180,360 and 720 days,respectively,in accordance with Latin square by double 7-order design.Silicon bead method was used to extract the DNA template that was further quantitatively detected using the real-time fluorescent quantitative PCR.Results：The DNA quantitative results varied a lot in the same kind of tissue sections preserved in dissimilar phase（P0.01）.No significant difference was seen in sections protected within one month,and yet,the DNA quantification was degraded significantly in those beyond 180 days.Also,the DNA content varied,to a certain extent,with diverse tissue sections kept for the same length（P0.01）,and the content was found relatively higher in sections of pulmonary,liver and kidney tissues.No significant difference was found concerning death ages（P0.6）.Conclusion：Forensic pathological tissue sections commonly exhibit lower content of DNA,i.e.,low copy number DNA,suggesting that the DNA nature is associated with the intervals of sections kept and tissue types.