血浆分离策略与提取方法对血浆DNA浓度的影响研究

Study on the influence of plasma separation strategies and extraction methods on plasma DNA concentration

目的:研究不同血浆分离策略与DNA提取方法对血浆DNA浓度的影响,以获得最适合用于血浆DNA检测分析的分离纯化方案。方法:收集20例健康人群外周血样本,进行血浆分离方案和DNA提取方法的比较。利用实时定量PCR技术检测不同离心速度和常见的3种DNA提取方法获得的血浆DNA浓度,同时比较提取方法对血浆DNA长短片段回收率的影响。t-检验分析各组间差异。选择最适合的血浆DNA分离策略对10例异常妊娠样本与12例健康妊娠样本中胎儿DNA进行检测与分析。结果:研究结果发现离心速度对血浆分离和DNA提取具有显著影响,3种离心策略获得的血浆DNA浓度,两两比较差异均具有统计学意义(P<0.05)。三步低速法、两步低速法和一步高速法获得的血浆DNA浓度分别为:(17.9±2.72)ng·mL^(-1)、(30.4±4.26)ng·mL^(-1)及(38.4±11.87)ng·mL^(-1)。除高速离心法外,样本冷冻对血浆分离结果无显著影响。三种常见DNA提取方法的研究发现,试剂盒法获得的血浆DNA浓度为(20.02±1.26)ng·mL^(-1),显著低于经典酚氯仿法(29.35±2.91)ng·mL^(-1)和碘化钠法(24.2±1.08)ng·mL^(-1)(P<0.05)。除酚氯仿法外,另两种提取法对血浆DNA长短片段的回收率差异有统计学意义(P<0.05),碘化钠法更适合短片段DNA的提取。利用两步低速法和碘化钠提取方法检测异常妊娠孕妇血浆DNA,结果发现子痫前期孕妇血浆中SRY基因浓度显著升高(P<0.05)。结论:两步低速离心法分离血浆与碘化钠法提取DNA可广泛应用于血浆DNA分离纯化及检测分析,子痫前期孕妇血浆中胎儿DNA浓度显著增高,有望用于异常妊娠的大规模筛查。

Objective:To investigate the effects of different plasma separation strategies and DNA extraction methods on plasma DNA concentration in order to obtain the most suitable plasma DNA separation and purification strategy.Methods:The peripheral blood samples of 20 healthy people were collected for comparison of plasma separation with DNA extraction method.The plasma DNA concentration obtained from different centrifugation speed of plasma separation and three commonly used DNA extraction methods was measured by real-time quantitative PCR.The effect of different extraction methods on the recovery of different length of DNA fragments was also studied and compared.The differences in different groups were analyzed by student's t-test.The most suitable plasma DNA separation strategy was selected to detect and analyze fetal DNA in 10 abnormal pregnancy samples and 12 healthy pregnancy samples.Results:The results showed that the centrifugation speed had significant effect on the plasma separation and DNA extraction.The difference of plasma DNA concentration obtained with the three DNA extraction methods was statistically significant(P<0.05).The plasma DNA concentration obtained by the three-step low speed method,the two-step low speed method and the one-step high speed method was(17.9±2.72)ng·mL^(-1),(30.4±4.26)ng·mL^(-1)L and(38.4±11.87)ng·mL^(-1)respectively.Sample freezing had no significant effect on plasma separation results except for high-speed centrifugation.The results of three DNA extraction methods showed that the plasma DNA concentration obtained by kit method(20.02±1.26)ng·mL^(-1)was significantly lower than that obtained by phenol chloroform method(29.35±2.91)ng·mL^(-1)and sodium iodide method(24.2±1.08)ng·mL^(-1)(P<0.05).Except for the phenol-chloroform method,the other two extraction methods also had significant differences in the DNA fragments recovery(P<0.05),and sodium iodide method was more suitable for the extraction of short DNA fragments.The detection results of plasma DNA from pregnant women with abnormal pregnancy showed that the concentration of SRY gene in the plasma of preeclampsia was significantly increased(P<0.05).Conclusion:The two-step low speed centrifugation method for plasma separation and sodium iodide method for DNA extraction can be widely used for plasma DNA separation,purification,detection and analysis.Fetal DNA concentration in the plasma of preeclampsia pregnant women increases significantly,which is expected to be used for abnormal pregnancy screening.