拭子提取黏膜部细菌DNA方法的优化

AN OPTIMIZED DNA EXTRATION PROTOCOL FOR THE BACTERIA IN MUCOUS MEMBRANES

目的:探索一种适合于黏膜部微生物组学分析的DNA抽提方法。方法:评价玻璃珠机械破碎法+溶菌酶裂解法结合DNA提取试剂盒法(Beads+Lysozyme+QIA法)和溶菌酶裂解法结合DNA提取试剂盒法(Lysozyme+QIA法)对9例鼻咽拭子和5株常见细菌的DNA提取效果。利用NanoDrop 2000测定DNA的浓度;以末端限制性片段长度多样性(T-RFLP)分析DNA的生态结构多态性。结果:两种方法对鼻咽拭子总DNA提取量无明显差别(P=0.288);T-RFLP结果显示,Beads+Lysozyme+QIA法可得到92种末段限制性片段(T-RF),高于Lysozyme+QIA法所得的43种,Beads+Lysozyme+QIA法SDI为(2.34±0.43)明显高于Lysozyme+QIA法(1.61±1.09)(P=0.03),Beads+Lysozyme+QIA法可以获得更丰富的TRF;Beads+Lysozyme+QIA法在表皮葡萄球菌、金黄色葡萄球菌、大肠埃希菌中可获得更高的DNA提取量(均P<0.05),在甲型溶血性链球菌和枯草芽孢杆菌中两种方法无明显差异(P>0.05)。结论:优化的拭子提取细菌DNA的方法可以更大程度地反映样本中微生物的多样性,减少偏倚;有利于黏膜部微生物组学的研究。

To explore a optimize DNA extraction protocol formucosae microbial genomics study.Methods.We evaluated the quality and the quantity of DNA, isolated by two protocols （bead beating assay ＋lysozyme treatment combined with DNA extraction kit and lysozyme treatment combined with DNA ex- traction kit） from nine human nasopharyngeal swabs and five strains of bacteria, using NanoDrop2000 UV- Vis Spectrophotometer. Results.Firstly, there was no significant difference in the quantity of DNA extraction of naspharyngeal swabs by those two methods. Secondly, T-RFLP showed that bead beating and lyso- zyme method can get much more T-RF fragments（92 fragments）, and it much more than the other method＇s （43 fragments）. For SDI, beads＋Lysozyme＋QIA group＇s （2.34±0.43） was higher than Lysozyme ±QIA group＇s （1.61±1.09, P = 0.03）. Additionally, thought the DNA extraction of alpha streptococcus and bacillus subtilis by beads＋Lysozyme＋QIA protocol was not difference between with the beads＋ Lyso- zyme＋QIA protocol＇s, more quantity of DNA extraction from Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli were extracted by beads ＋ Lysozyme ＋ QIA method. Conclusion： The improved Bead beating assay ＋ lysozyme treatment combined with DNA extraction method can get more DNA ploymorphism, and its low bias characteristic, so it is a sutiable protocl for mucous microflora met- agenomics studies.