溃疡性结肠炎相关肠道菌群荧光定量PCR检测方法的建立
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摘要
目的:为了进一步探索菌群失调对溃疡性结肠炎(Ulcerative Colitis,UC)的影响,建立与UC相关的肠道菌群荧光定量qRT-PCR检测方法。方法:通过筛选确定7种与UC发病及病程进展有较强相关性的肠道微生物种类,设计PCR引物,扩增出标志性片段重组到质粒中作为标准品,利用实时荧光定量PCR技术建立相关肠道菌的定量检测方法。结果:成功建立了检测双歧杆菌属等优势菌群、大肠埃希菌等条件致病菌及肠道总菌群的qRT-PCR方法,各相关菌种重组质粒建立的标准曲线相关系数≥0.998。结论:通过实验验证筛选了7种与UC相关的肠道菌群,并建立其绝对定量的qRT-PCR检定方法,具有特异性及准确度高等特点。
Objective: To establish a quantitative real-time PCR method for detecting gut bacteria in patients with ulcerative colitis in order to further explore the effects of imbalanced gut flora on ulcerative colitis(UC). Methods: We identified 7 gut microbes that were strongly associated with the pathogenesis of UC. Specific PCR primers were designed to construct a standard recombinant plasmid containing specific amplified fragments. The absolute quantitative method of real-time PCR was established by using recombinant plasmids as standards. Results: The real-time PCR method for the detection of such target bacteria as Bifidobacterium, Escherichia coli, and total intestinal flora, was successfully established. The standard curve of gut bacteria showed a good linear relation with correlation coefficient ≥0.998. Conclusion: The SYBR Green real-time PCR method can detect seven intestinal bacteria in ulcerative colitis specifically and provide a convenient and effective method for exploring the correlation between the imbalance of gut bacteria and UC pathogenesis.
Objective: To establish a quantitative real-time PCR method for detecting gut bacteria in patients with ulcerative colitis in order to further explore the effects of imbalanced gut flora on ulcerative colitis(UC). Methods: We identified 7 gut microbes that were strongly associated with the pathogenesis of UC. Specific PCR primers were designed to construct a standard recombinant plasmid containing specific amplified fragments. The absolute quantitative method of real-time PCR was established by using recombinant plasmids as standards. Results: The real-time PCR method for the detection of such target bacteria as Bifidobacterium, Escherichia coli, and total intestinal flora, was successfully established. The standard curve of gut bacteria showed a good linear relation with correlation coefficient ≥0.998. Conclusion: The SYBR Green real-time PCR method can detect seven intestinal bacteria in ulcerative colitis specifically and provide a convenient and effective method for exploring the correlation between the imbalance of gut bacteria and UC pathogenesis.
引文
[1]闫爽.美洲大蠊提取物对大鼠溃疡性结肠炎肠道菌群的影响及昆虫源提取物抗肿瘤活性机制初步研究[D].大理:大理大学,2017.
[2]MA X W,HU Y C,LI X,et al.Periplaneta americana Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Rats by Keap1/Nrf-2 Activation,Intestinal Barrier Function,and Gut Microbiota Regulation[J].Frontiers in Pharmacology,2018,9:1-16.
[3]王朋川,何苗,张汉超,等.康复新液对2,4-二硝基氯苯联合醋酸诱导大鼠溃疡性结肠炎的影响[J].大理大学学报,2016,1(4):17-21.
[4]李静,陈卫刚.肠道菌群治疗溃疡性结肠炎的研究进展[J].实用医学杂志,2017,33(24):4189-4192.
[5]程悦,左浩江,廖虹瑜,等.肠道常见菌群荧光定量PCR检测方法的建立[J].现代预防医学,2014,41(23):4338-4341.
[6]李超男.美洲大蠊提取物对溃疡性结肠炎大鼠模型肠道菌群影响的初步研究[D].大理:大理大学,2016.
[7]王婷婷.肠道菌群结构变化与结直肠癌发生发展关系的研究[D].上海:上海交通大学,2012.
[8]RINTTILA T,KASSINEN A,MALINEN E,et al.Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR[J].Journal of Applied Microbiology,2004,97(6):1166-1177.
[9]NELSON E A,PALOMBO E A,KNOWLES S R.Comparison of methods for the extraction of bacterial DNA from human faecal samples for analysis by real-time PCR[J].Current Research,Technology and Education Topics in Applied Microbiology and Microbial Biotechnology,2010,2:1479-1485.
[10]LOUIS P,FLINT H J.Development of a semiquantitative degenerate real-time PCR-based assay for estimation of numbers of butyryl-coenzyme A(CoA)CoA transferase genes incomplex bacterial samples[J].Applied&Environmental Microbiology,2007,73(6):2009-2012.
[11]DEVKOTA S,WANG Y,MUSCH M W,et al.Dietary-fatinduced taurocholic acid promotes pathobiont expansion and colitis in Il10-/-mice[J].Nature,2012,487(7405):104-108.
[12]赵洁,马晨,席晓敏,等.实时荧光定量PCR技术在肠道微生物领域中的研究进展[J].生物技术通报,2014(12):61-66.
[13]周正华.肠道微环境与溃疡性结肠炎[J].世界华人消化杂志,2016,24(11):1695-1700.
[14]DELROISSE J M,BOULVIN A L,PARMENTIER I,et al.Quantification of Bifidobacterium spp.and Lactobacillus spp.in rat fecal samples by real-time PCR[J].Microbiological Research,2006,163(6):663-670.
[15]王丽娜,周旭春.肠道菌群与肠黏膜免疫及相关肠道疾病的研究进展[J].中国微生态学杂志,2017,29(4):494-496.
[16]杜若曦,郭明璋,谢子鑫,等.合成生物学在改善肠道健康状态中的应用与展望[J].生物技术通报,2018,34(1):49-59.
[17]WENHUA Z,JUN L,BENYAN W.Gene expression profiling of the mouse gut:Effect of intestinal flora on intestinal health[J].Molecular Medicine Reports,2017,17(3):3667-3673.
[18]李东萍,郭明璋,许文涛.16S rRNA测序技术在肠道微生物中的应用研究进展[J].生物技术通报,2015,31(2):71-77.
[19]陈军,李慧玲,董建一,等.标准品制备方法对FQ-PCR定量基因表达水平的影响[J].中国生物化学与分子生物学报,2012,28(11):1064-1068.
[20]高晓琳,贾瑞贞,谢亮,等.不同双歧杆菌标准品制备法的比较研究[J].四川大学学报(医学版),2016,47(4):605-608.
[2]MA X W,HU Y C,LI X,et al.Periplaneta americana Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Rats by Keap1/Nrf-2 Activation,Intestinal Barrier Function,and Gut Microbiota Regulation[J].Frontiers in Pharmacology,2018,9:1-16.
[3]王朋川,何苗,张汉超,等.康复新液对2,4-二硝基氯苯联合醋酸诱导大鼠溃疡性结肠炎的影响[J].大理大学学报,2016,1(4):17-21.
[4]李静,陈卫刚.肠道菌群治疗溃疡性结肠炎的研究进展[J].实用医学杂志,2017,33(24):4189-4192.
[5]程悦,左浩江,廖虹瑜,等.肠道常见菌群荧光定量PCR检测方法的建立[J].现代预防医学,2014,41(23):4338-4341.
[6]李超男.美洲大蠊提取物对溃疡性结肠炎大鼠模型肠道菌群影响的初步研究[D].大理:大理大学,2016.
[7]王婷婷.肠道菌群结构变化与结直肠癌发生发展关系的研究[D].上海:上海交通大学,2012.
[8]RINTTILA T,KASSINEN A,MALINEN E,et al.Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR[J].Journal of Applied Microbiology,2004,97(6):1166-1177.
[9]NELSON E A,PALOMBO E A,KNOWLES S R.Comparison of methods for the extraction of bacterial DNA from human faecal samples for analysis by real-time PCR[J].Current Research,Technology and Education Topics in Applied Microbiology and Microbial Biotechnology,2010,2:1479-1485.
[10]LOUIS P,FLINT H J.Development of a semiquantitative degenerate real-time PCR-based assay for estimation of numbers of butyryl-coenzyme A(CoA)CoA transferase genes incomplex bacterial samples[J].Applied&Environmental Microbiology,2007,73(6):2009-2012.
[11]DEVKOTA S,WANG Y,MUSCH M W,et al.Dietary-fatinduced taurocholic acid promotes pathobiont expansion and colitis in Il10-/-mice[J].Nature,2012,487(7405):104-108.
[12]赵洁,马晨,席晓敏,等.实时荧光定量PCR技术在肠道微生物领域中的研究进展[J].生物技术通报,2014(12):61-66.
[13]周正华.肠道微环境与溃疡性结肠炎[J].世界华人消化杂志,2016,24(11):1695-1700.
[14]DELROISSE J M,BOULVIN A L,PARMENTIER I,et al.Quantification of Bifidobacterium spp.and Lactobacillus spp.in rat fecal samples by real-time PCR[J].Microbiological Research,2006,163(6):663-670.
[15]王丽娜,周旭春.肠道菌群与肠黏膜免疫及相关肠道疾病的研究进展[J].中国微生态学杂志,2017,29(4):494-496.
[16]杜若曦,郭明璋,谢子鑫,等.合成生物学在改善肠道健康状态中的应用与展望[J].生物技术通报,2018,34(1):49-59.
[17]WENHUA Z,JUN L,BENYAN W.Gene expression profiling of the mouse gut:Effect of intestinal flora on intestinal health[J].Molecular Medicine Reports,2017,17(3):3667-3673.
[18]李东萍,郭明璋,许文涛.16S rRNA测序技术在肠道微生物中的应用研究进展[J].生物技术通报,2015,31(2):71-77.
[19]陈军,李慧玲,董建一,等.标准品制备方法对FQ-PCR定量基因表达水平的影响[J].中国生物化学与分子生物学报,2012,28(11):1064-1068.
[20]高晓琳,贾瑞贞,谢亮,等.不同双歧杆菌标准品制备法的比较研究[J].四川大学学报(医学版),2016,47(4):605-608.