国产血浆游离DNA提取试剂盒的性能评价

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TaqMan 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，r2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。

Performance evaluation of the domestic cell-free DNA extraction kit

Objective: To evaluate the domestic kit of cell free DNA(cfDNA) extraction from plasma. Methods: Plasma pool A was composed of volunteers plasma, and different length of DNAs were added into the plasma pool A to form the plasma pool B1-B4, the addition of DNA fragments length were 102, 268 and 402 bp and their mixture, respectively.The cfDNA in plasma pool A and pool B1-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 and the amount of plasma 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 B1-B4 of two Extraction Kits were determined by the nucleic acid analyzer. Results: The Ct values of cfDNA in plasma pool A extracted by Qiagen and domestic kits 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 linear correlations between the cfDNA concentrations extracted by Qiagen and domestic Kit and the plasma pool A dosage, r2 were 0.979(P<0.01) and 0.963(P<0.01), respectively; Gel electrophoresis results showed that the cfDNA in plasma pool A extracted by the two methods appeared obvious bands at 102, 268 and 402 bp, and 1 204 bp fragments did not appear. The recovery rates of 102, 268， 402 bp and their mixture in the plasma pool B1-B4 extracted by Qiagen and domestic kits were 64.81%±4.27%, 80.40%±4.31%, 88.40%±6.11%, 83.50%±5.21% and 73.50%±5.33%, 85.20%±4.49%, 89.30%±5.91%, 85.64%±4.48%, respectively, and there were significant difference between two methods in the recovery rates of 102 bp and 268 bp DNA fragments(t=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 402 bp 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.