TaqMan实时荧光定量PCR检测肠道细菌Akkermansia Muciniphila
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摘要
目的建立Akkermansia muciniphila(A.muciniphila)TaqMan实时荧光定量PCR检测方法,实现粪便中A.muciniphila的定量检测。方法根据GenBank公布的A.muciniphila 16S rRNA基因高保守序列,设计合成特异引物和TaqMan探针,构建重组质粒作为标准品,通过反应体系和反应条件的优化,绘制标准曲线,同时将建立的方法用于10份成年人粪便标本的检测。结果 TaqMan实时荧光定量PCR检测A.muciniphila,仅需1.5h,该方法线性好,相关系数R~2=0.9991,扩增效率E=94.7%;重复性好,组内和组间变异系数均小于3%;灵敏度可达到5×10~3拷贝/μl;特异性良好。10份成年人粪便标本共检出9份为阳性,阳性粪便标本中A.muciniphila平均含量为2.70×10~8cells/g粪便。结论本研究建立的TaqMan实时荧光定量PCR能够快速、灵敏、特异地定量检测粪便标本中A.muciniphila。
Objective To establish a TaqMan quantitative real-time PCR assay for the detection of Akkermansia Muciniphila,and to quantitatively detect A.muciniphila in stools.Methods Specific primers and TaqMan probe were designed and the rapid real-time PCR assay was established and optimized based on the conservative sequence of 16 S rRNA gene of A.Muciniphila published on GenBank.Then the recombinant plasmids containing a part of A.muciniphila 16 S rRNA sequence were constructed to draw the curves through optimizing the reaction system and condition.Ten faecal specimens of adults were detected by this method.Results The TaqMan quantitative real-time PCR assay could be finished in 1.5 hours.The correlation coefficient of the quantitative standard curve was 0.9991,and the amplification efficiency was 94.7%.The assay had a good reproducibility,and its variation coefficients within and between groups were all less than 3%.The sensitivity of the assay was 5×103 copies per μl,with high specificity.A total of 9 faecal specimens from 10 adults were positive and the average level of A.muciniphila was 2.70×108 cells/g faeces.Conclusion The TaqMan quantitative real-time PCR assay is rapid,sensitive and specific to detect A.muciniphila from stools.
Objective To establish a TaqMan quantitative real-time PCR assay for the detection of Akkermansia Muciniphila,and to quantitatively detect A.muciniphila in stools.Methods Specific primers and TaqMan probe were designed and the rapid real-time PCR assay was established and optimized based on the conservative sequence of 16 S rRNA gene of A.Muciniphila published on GenBank.Then the recombinant plasmids containing a part of A.muciniphila 16 S rRNA sequence were constructed to draw the curves through optimizing the reaction system and condition.Ten faecal specimens of adults were detected by this method.Results The TaqMan quantitative real-time PCR assay could be finished in 1.5 hours.The correlation coefficient of the quantitative standard curve was 0.9991,and the amplification efficiency was 94.7%.The assay had a good reproducibility,and its variation coefficients within and between groups were all less than 3%.The sensitivity of the assay was 5×103 copies per μl,with high specificity.A total of 9 faecal specimens from 10 adults were positive and the average level of A.muciniphila was 2.70×108 cells/g faeces.Conclusion The TaqMan quantitative real-time PCR assay is rapid,sensitive and specific to detect A.muciniphila from stools.
引文
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[3] Karlsson CL,Onnerf?lt J,Xu J,et al.The microbiota of the gut in preschool children with normal and e×cessive body weight[J].Obesity,2012,20(11):2257-2261.
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[6] Zhong Y,Marungruang N,F?k F,et al.Effects of two whole-grain barley varieties on caecal SCFA,gut microbiota and plasma inflammatory markers in rats consuming low-and high-fat diets[J].The British Journal of Nutrition,2015,113(10):1558-1570.
[7] Qin J,Li Y,Cai Z,et al.A metagenome-wide association study of gut microbiota in type 2 diabetes[J].Nature,2012,490(7418):55-60.
[8] Ganesh BP,Klopfleisch R,Loh G,et al.Commensal akkermansia muciniphila e×acerbates gut inflammation in salmonella typhimurium-infected gnotobiotic mice[J].PLOS One,2013,8(9):e74963.
[9] Png CW,Lindén SK,Gilshenan KS,et al.Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria[J].The American Journal of Gastroenterology,2010,105(11):2420-2428.
[10] Collado MC,Derrien M,Isolauri E.Intestinal integrity and Akkermansia muciniphila,a mucin-degrading member of the intestinal microbiota present in infants,adults,and the elderly[J].Applied and Environmental Microbiology,2007,73(23):7767-7770.
[2] Everard A,Belzer C,Geurts L,et al.Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(22):9066-9071.
[3] Karlsson CL,Onnerf?lt J,Xu J,et al.The microbiota of the gut in preschool children with normal and e×cessive body weight[J].Obesity,2012,20(11):2257-2261.
[4] Zhang X,Shen D,Fang Z,et al.Human gut microbiota changes reveal the progression of glucose intolerance[J].PLOS One,2013,8(8):e71108.
[5] Swidsinski A,D?rffel Y,Loening-Baucke V,et al.Acute appendicitis is characterised by local invasion with Fusobacterium nucleatum/necrophorum[J].Gut,2011,60(1):34-40.
[6] Zhong Y,Marungruang N,F?k F,et al.Effects of two whole-grain barley varieties on caecal SCFA,gut microbiota and plasma inflammatory markers in rats consuming low-and high-fat diets[J].The British Journal of Nutrition,2015,113(10):1558-1570.
[7] Qin J,Li Y,Cai Z,et al.A metagenome-wide association study of gut microbiota in type 2 diabetes[J].Nature,2012,490(7418):55-60.
[8] Ganesh BP,Klopfleisch R,Loh G,et al.Commensal akkermansia muciniphila e×acerbates gut inflammation in salmonella typhimurium-infected gnotobiotic mice[J].PLOS One,2013,8(9):e74963.
[9] Png CW,Lindén SK,Gilshenan KS,et al.Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria[J].The American Journal of Gastroenterology,2010,105(11):2420-2428.
[10] Collado MC,Derrien M,Isolauri E.Intestinal integrity and Akkermansia muciniphila,a mucin-degrading member of the intestinal microbiota present in infants,adults,and the elderly[J].Applied and Environmental Microbiology,2007,73(23):7767-7770.