摘要
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 explore the influence factors of DNA extraction from human intestinal flora.Methods DNA was extracted from intestinal flora samples by using lysozyme and SDS ＋ quartz sand and phenol chloroform extraction.16 SrDNA was amplified by PCR.The changes in concentration and purity of intestinal bacterial DNA were compared among different sample amounts,temperatures and time intervals;high-throughput sequencing results were tested by Chao index.Results The concentration of bacterial DNA extracted using 20 mg of feces was the highest.The concentration of extracted DNA began to decrease after standing 3hours at room temperature,12 hours at-20℃,and 48 hours at-70℃;The purity of samples were above 1.8.The Chao index curves became smooth,indicating that the amount of sequencing data was large enough.Conclusion The appropriate amount of fecal sample for extraction of bacterial genomic DNA is20 mg and the samples should not be placed at room temperature for more than 2hours.The concentration of DNA extracted with the methods used in our study is up to the requirement of high throughput sequencing.
出处
《中国微生态学杂志》
CAS
CSCD
2016年第3期271-274,279,共5页
Chinese Journal of Microecology
关键词
人
粪便
细菌基因组
DNA提取
影响因素
Human
Feces
Bacterial genome
DNA extraction
Influencing factors
