国产血浆游离DNA提取试剂盒的性能评价

Performance evaluation of the domestic cell-free DNA extraction kit

目的:对国产血浆游离DNA(cell-free DNA,cfDNA)提取试剂盒进行评价。方法:用志愿者血浆构成血浆池A,再分别加入固定量片段长度为102、268、402 bp和所有片段混合物的DNA,形成血浆池B1-B4。用Qiagen和国产柱式法cfDNA提取试剂盒分别提取血浆池A和血浆池B1-B4中的cfDNA。用Taq Man qPCR分析两种试剂盒提取血浆池A中cfDNA的提取效率,同时分析cfDNA浓度与血浆用量之间的关系,用琼脂糖凝胶电泳分析提取的cfDNA的片段完整性。核酸测定仪分析两种试剂盒对血浆池B1-B4中不同DNA片段的回收率。结果:Qiagen和国产试剂盒提取的血浆池A中cfDNA的Ct值分别为27.94±0.423和27.41±0.356,国产试剂盒提取效率明显高于Qiagen试剂盒(t=4.29,P<0.01);琼脂糖凝胶电泳结果表明,两种方法提取的血浆池A中cfDNA在102、268和402 bp处均出现明显条带,而1 204 bp片段未出现。Qiagen和国产试剂盒提取的cfDNA浓度与血浆池A的血浆用量之间均存在明显的线性相关,r^2分别为0.979(P<0.01)和0.963(P<0.01)。Qiagen和国产试剂盒提取的血浆池B1-B4血浆中102、268、402 bp及其混合物的回收率分别为64.81%±4.27%,80.40%±4.31%,88.40%±6.11%,83.50%±5.21%和73.50%±5.33%,85.20%±4.49%,89.30%±5.91%,85.64%±4.48%,两者在102和268 bp片段的回收率上差异显著(t=5.69,P<0.01;t=3.44,P<0.01),而在402 bp及混合物的回收率上无明显差异(t=0.47,P>0.05;t=1.39,P>0.05)。结论:国产cfDNA提取试剂盒在各项评价性能上均可与Qiagen产品媲美。

Objective:To evaluate the domestickit of cell-free DNA(cfDNA)extraction from plasma.Methods:Plasma pool A was composed of volunteers plasma,and d ifferent length of DNAs were added in to the plasma pool A to form the plasma pool Bl-B4,the addition of DNA fragments length were 102,268 and 402 bp and their mixture,respectively.The cfDNA in plasma pool Al-B4 were extracted by Qiagen and domestic column free DNA extraction kit.The extraction efficiencies of cfDNA in plasma pool A of two Extraction Kits were analyzed by Ct value of TaqMan qPCR,and the linear relationships between the extracted cfDNA concentrations of two Extraction Kits a pool A were analyzed,and fragment size bias of cfDNA were analyzed by gel electrophoresis.The different DNA fragments recovery rates in the plasma pool Bl-B4 of two Extraction Kits were determ ined by the nucleic acid analyzer.Results:The Ct values of cfDNA in plasma pool A extracted by Qiagen and domestick its were 27.94±0.423 and 27.41±0.356,respectively,and the cfDNA extraction efficiency of domestic kit was obviously higher than Qiagen kit(t=4.29,P<0.01);There were a significant line-ar correlations between the cfDNA concentrations extracted by Qiagen and domestic K it and the plasma pool A dosage,r2 were 0.979(P<0.01)and 0.963(P<0.01),resp e ctive ly;G el electrophoresis results showed that the cfD N A in plasm a pool A extracted by the two m ethods appeared obvious bands at 102,268 and 402 b p,and 1 204 bp fragm ents d id not appear.The recovery rates o f 102,268,402 bp and th e ir m ixture in the plasmapool B1-B4 extracted by Qiagen and domestic its were 64.8 1%士4.2 7%,80.40%±4.3 1%,88.40%±6.1 1%,83.50%±5.2 1%and 73.5 0%±5.3 3%,85.20%士4.49%,89.30%±5.91%,85.64%±4.48%,respectively,and there were significant difference between two methods in the recovery rates of 102bp and 268bp DNA fra gments=5.69,P<0.01;t=3.44,P<0.01),then there were no significant difference between two methods in the recovery rates of 402bp and DNA mixture(t=0.47,P>0.05;t=1.39,P>0.05).Conclusion;Domestic cfDNA extraction kit can be comparable with Qiagen product in all evaluation parameters.