慢性阻塞性肺疾病患者支气管肺泡灌洗液细菌宏基因组DNA的制备

Preparation of metagenomic DNA from bronchoalveolar lavage fluids of patients with chronic obstructive pulmonary diseases

【目的】优化稳定期慢性阻塞性肺疾病(chronic obstructive pulmonary diseases,COPD)患者支气管肺泡灌洗液(bronchoalveolar lavage fluids,BALF)细菌宏基因组DNA的提取方法,以便于高效提取微量的细菌DNA进行后续的PCR反应和测序。【方法】取稳定期COPD患者的BALF 5mL,离心收集细胞。为了有效提取样品中革兰氏阳性菌的基因组,对QIAGEN的DNA提取试剂盒的操作步骤进行优化:加入裂解缓冲液ATL后首先运用研磨珠和多功能生物样品匀质器破碎菌壁,再加入蛋白酶K孵育,然后加入裂解缓冲液AL振荡混匀。无水乙醇沉淀DNA后,将全部溶液过柱,用洗液AW1和AW2各洗柱一次,最后加50μL洗脱液洗脱DNA。提取的DNA定量后,运用PCR方法检测样本中的细菌16S rDNA量,并按照测序要求构建DNA文库进一步验证。【结果】试剂盒优化法提取的BALF的DNA总量为467.5(135.0-1697.5)ng,明显高于按照传统酚-氯仿法提取的DNA总量95.0(0-612.5)ng,并且所提取的DNA可以很好的扩增细菌的16S rDNA以及构建DNA文库,改良后的扩增产物明显增多(P=0.002)。【结论】使用DNA提取试剂盒结合研磨珠和多功能生物样品匀质器破菌壁的方法能够更高效的提取BALF中微量的宏基因组DNA,为进一步的测序和菌群分析打下基础。

[ Objective ] To optimize the method of isolating a small amount of metagenomic DNA efficiently from bronchoalveolar lavage fluids （BALF） of patients with stable chronic obstructive pulmonary diseases （COPD） , which will facilitate subsequent PCR and DNA sequencing. [ Methods] BALF （SmL） of stable COPD patients was spun down to collect the cells. To extract genomic DNA from Gram-positive bacteria more efficiently, QIAGEN＇s DNA extraction protocol was optimized as follows： Added Buffer ATL to the pellets and used bead tubes and tissue homogenizers to break cell walls; then added proteinase K and incubated; after adding Buffer AL and ethanol, pipetted the mixture into a DNeasy spin column then centrifuged; washed the column with Buffer AWl and Buffer AW2, finally added 50μL Buffer AE to elute DNA. After measuring the total DNA concentration, the bacterial 16S rDNA was amplified by PCR and amplicon libraries were created for further determination. [ Results] The DNA content of BALF with optimized protocols was 467.5 （135.0-1697.5） ng, which was significantly higher than those extracted with phenol-chloroform 95.0 （0- 612.5） ng. After optimizing, more 16S rDNA PCR production can be obtained for future analysis （P = 0.002）. [ Conclusion] The optimized DNA extraction methods combining DNA isolation kits with bead-beating were more efficient in isolating tiny metagenomic DNA from BALF.