甲醛固定石蜡包埋组织4种DNA提取方法的比较分析

A Comparative Analysis of Four Different DNA Extraction Methods from Formalin-fixed Paraffin-embedded Tissue in Forensic Medicine

本研究以10例甲醛固定石蜡包埋组织(FFPET)作为疑难检材,将其制成蜡膜,分别以二甲苯-Chelex100法、脱蜡液-Chelex100法、脱蜡液-Vazyme FastPure FFPE DNA Isolation试剂盒(简称FP试剂盒)、脱蜡液-北京天根石蜡包埋组织基因组提取试剂盒法(简称天根试剂盒)提取DNA,进行紫外分光光度计测定(浓度、纯度)及短串联重复序列(STR)分型检测(23个常染色体),通过比较分析FFPET在4种不同提取方法中的提取质量、STR分型质量和检出率,从而寻求一种高效简便、经济实用、方便快捷的甲醛固定石蜡包埋组织中DNA提取方法。结果表明:应用4种方法提取的石蜡包埋组织蜡膜DNA经紫外分光光度计测定后显示,应用2种Chelex100法提取的DNA浓度高于2款试剂盒,试剂盒提取的DNA的纯度优于另外2种方法,以上差异均具有统计学意义(P<0.05);FP试剂盒提取DNA的平均浓度和纯度均优于天根试剂盒,无显著性差异(P>0.05);STR分型普遍存在不同基因座和同一基因座不同等位基因之间的扩增不均衡,呈现前高后低状态;STR平均检出率为FP试剂盒>天根试剂盒>Chelex100法,2种Chelex100法STR基因座检出率比较无显著差异(P>0.05),但分别和另外2种方法试剂盒方法相比有显著差异(P<0.05),FP试剂盒和天根试剂盒法相比有显著差异(P<0.05)。因此,在涉及甲醛固定石蜡包埋组织这类疑难检材的法医DNA鉴定案件时,脱蜡液-Vazyme FastPure FFPE DNA Isolation试剂盒法优于其余3种提取DNA方法。

The study used myocardial tissues of 10 forensic FFPET patients and the samples were prepared into films.The DNA was extracted using xylene-Chelex100 method,dewaxing solution-Chelex100 method,dewaxing solution-Vazyme Fast-Pure FFPE DNA Isolation kit(abbreviated as FP kit)and dewaxing solution-Beijing Tiangen paraffin-embedded tissue genome extraction kit(abbreviated as Tiangen kit),and determined by ultraviolet spectrophotometer for concentration and purity,and short tandem repeats(STR)typing test(23 autosomes).The extraction quality,STR typing quality and detection rate of FFPET in four different extraction methods were compared and analyzed to find an efficient,simple,economical,practical,and convenient method.The results showed that for the quality of extrated DNA,the concentration of DNA extracted by two Chelex100 methods were higher than the two test kits,and the purity of DNA extracted by the kits were superior to that of the other two methods.The differences were statistically significant(P<0.05).The average concentration and purity of DNA extracted by FP kit were superior to those by Tiangen kit without significant differences(P>0.05).For the quality of STR typing and detection rate,STR typing generally shows the imbalance of amplification between different loci and different alleles of the same locus,with the former being higher and the later lower.The average detection rate of STR was in the order of:FP kit>Tiangen kit>Chelex100 method.There were no significant difference in the detection rates of STR loci between the two Chelex100 methods(P>0.05),whereas there were significant differences between them and the other two methods(P<0.05).There was a significant difference between the FP kit and Tiangen kit(P<0.05).Therefore when dealing with difficult samples such as formaldehyde-fixed paraffin-embedded tissues in forensic DNA identification cases,the dewaxing solution-Vazyme FastPure FFPE DNA Isolation kit was superior to the other three DNA extraction methods.