摘要
目的分别应用两种方法进行血浆游离胎儿DNA的提取,判定及筛选最佳的血浆游离胎儿DNA的提取方法,为后续试验提取游离胎儿DNA的方法提供参考依据。方法收集30例健康妊娠孕妇血浆,用离心柱法和磁珠法进行血浆游离DNA的提取,经甲基化敏感限制性内切酶Bst UI酶切后使用微量紫外分光光度计测量所提取的游离胎儿DNA的含量及纯度,再使用实时荧光定量PCR扩增方法测定扩增效率,进行统计学分析。结果离心柱法所提取的游离胎儿DNA的测定结果为:含量(43. 27±21. 01) ng/μl;纯度A260/A280 (0. 999 7±0. 084 42);扩增效率为94%;而磁珠法提取的游离胎儿DNA的测定结果为:含量(31. 74±12. 25) ng/μl:纯度A260/A280 (0. 878 3±0. 052 66);扩增效率为87%。两组方法提取游离DNA的浓度、纯度比较,差异有统计学意义(均P<0. 05)。结论离心柱法比磁珠法操作更简便、快捷。经测定提取的DNA的浓度及纯度,扩增效率后可判断离心柱法所提取的游离胎儿DNA的浓度、纯度及扩增效率都远远优于磁珠法;离心柱法比磁珠法更适用于后续无创产前诊断研究试验。
Objective To compare with two methods of extracting free fetal DNA in maternal plasma, determine which method is more optimal for the extraction of cell-free fetal DNA procedure. Methods The centrifugal column method and the magnetic beads method were used to extract free fetal DNA in maternal plasma. After the digestion of the methylation sensitive restriction enzyme BstUI enzyme, trace ul- traviolet spectrophotometer were used to measure the content and purity of fi＇ee fetal DNA. Then the real-time fluorescent quantitative PCR was used to detect the amplification efficiency. The statistical analysis was made. Results The determination results of the centrifugal column method were listed as following： the content ： 43.27 ±21.01 ng/μl： ; A260 / A280 ： 1.00±0. 08 ; amplification efficiency was 94% ; while the determination results of the magnetic beads method as following： content： 31.74 ±12. 25 ng/μl; A260 / A280： 0. 88± 0. 05 ; amplification efficiency was 87%. There was significant difference between the two methods. （ P〈0.05 ） . Conclusion The centrifugal column method operates is more simple and convenient than the magnetic bead method, which is more suitable for the subsequent non-prenatal diagnosis research.
作者
冉贵萍
赵海鸥
谢艳平
陈英
钟贞强
RAN Gui-Ping;ZHAO Hai-Ou;XIE Yan-Ping(Shanghai Fengcheng Hospital,the Ninth People＇s Hospital Affiliated to Shanghai Jiaotong University School of Medicine,Shanghai,201411,China)
出处
《中国妇幼保健》
CAS
2018年第21期4948-4950,共3页
Maternal and Child Health Care of China
基金
上海市奉贤区科委项目(项目编号:奉(科)20151007)
