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目的研究提取人类粪便中细菌基因组DNA的影响因素。方法采用溶菌酶和十二烷基磺酸钠(SDS)+石英砂+酚-氯仿抽提法提取粪便标本中细菌DNA,用PCR扩增细菌16SrDNA。比较不同粪便量,不同放置时间,不同放置温度下的粪便细菌DNA浓度及纯度改变;用Chao index评测高通量测序的结果。结果采用20mg粪便量提取出的细菌DNA的浓度最高;常温下放置3h后,提取的细菌DNA的浓度开始下降;在-20℃放置12h后,细菌DNA的浓度开始下降;-70℃放置48h后细菌DNA的浓度开始下降;样品纯度均在1.8以上;Chao index曲线趋于平缓表明测序数据量足够大。结论提取肠道细菌基因组DNA时,粪便标本取样量以20mg为宜,常温下粪便标本放置不超过2h,本研究所使用的方法所提取的DNA浓度可以达到高通量测序的样本要求。
Objective To study the factors affecting the extraction of bacterial genomic DNA in human feces. Methods Bacterial DNA was extracted from stool samples by lysozyme and sodium dodecyl sulfate (SDS) + quartz sand + phenol-chloroform extraction. The bacterial 16S rDNA was amplified by PCR. The fecal bacterial DNA concentration and purity at different storage times, storage times and different storage temperatures were compared. The results of high-throughput sequencing were evaluated by Chao index. Results The concentration of bacterial DNA extracted by 20mg feces was the highest. After 3h at room temperature, the concentration of bacterial DNA began to decrease. After 12h at -20 ℃, the concentration of bacterial DNA began to decrease. After 48h at -70 ℃, The concentration of bacterial DNA began to decline; the purity of the samples was above 1.8; the Chao index curve tended to show that the amount of sequencing data was large enough. Conclusions When extracting genomic DNA of intestinal bacteria, the amount of stool sample should be 20mg, and the stool sample should be kept at room temperature for no more than 2h. The DNA concentration extracted by this method can meet the high-throughput sequencing sample requirements.