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

发布时间：2018-12-14 12:42

【摘要】：【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。
[Abstract]:[objective] to optimize the extraction method of bacterial macro genomic DNA from bronchoalveolar lavage fluid (bronchoalveolar lavage fluids,BALF) from patients with stable chronic obstructive pulmonary disease (chronic obstructive pulmonary diseases,COPD). It was convenient to extract microamounts of bacterial DNA for subsequent PCR reaction and sequencing. [methods] the BALF 5mL of stable COPD patients was collected and the cells were collected by centrifugation. In order to extract the genome of Gram-positive bacteria from the sample effectively, the operating procedure of DNA extraction kit of QIAGEN was optimized. After adding ATL, the bacteria wall was first broken by grinding beads and multifunctional biological sample homogenizer. After incubation with protease K, AL was added to the lytic buffer. After precipitating DNA with anhydrous ethanol, the solution was crossed over the column, then washed with AW1 and AW2, and then the DNA. was eluted with 50 渭 L eluant. After quantifying the extracted DNA, the amount of 16s rDNA in the sample was detected by PCR method, and the DNA library was constructed according to the requirements of sequencing. [results] the total DNA of BALF extracted by the optimization method of the kit was 467.5 (135.0-1697.5) ng,. The total amount of DNA extracted by traditional phenol-chloroform method was 95.0 (0-612.5) ng, and the extracted DNA could amplify 16s rDNA of bacteria and construct DNA library. (P0. 002). [conclusion] the method of DNA extraction kit combined with grinding beads and multifunctional biological sample homogenizer can be used to extract microamounts of macro genomic DNA, from BALF more efficiently. It will lay a foundation for further sequencing and microflora analysis.
【作者单位】：北京大学第三医院呼吸科;北京大学医学部-密歇根大学医学院转化医学与临床研究联合研究所;密西根大学医学院;
【基金】：北京大学医学部-密歇根大学医学院转化医学与临床研究联合研究所,转化医学-密西根项目(BMU20110176)~~
【分类号】：R563.9

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