A semi-automated protocol for Archaea DNA extraction from stools

BMC Research Notes

Michel Drancourt¹  Marielle Bedotto Buffet²  Pierre-Yves Ramonet²  Saber Khelaifia² 

[1] Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin, Cedex 5, Marseille, France;Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005, Marseille, France

关键词: Archaeal DNA;    DNA extraction;    Microbiota;    Methanogenic archaea;    Human-associated archaea;   

Others  :  1142759
DOI  :  10.1186/1756-0500-6-186

 received in 2012-09-20, accepted in 2013-04-17,  发布年份 2013

【 摘 要 】

Background

The PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens.

Findings

We tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1® DNA Tissue Kit (Qiagen, Courtaboeuf, France) yielded 35/110 (32%) positives for the real-time PCR detection of the Methanobrevibacter smithii 16S rRNA gene, with average Ct values of 36.1. A semi-automated protocol combining glass-powder crushing, overnight proteinase K digestion and lysis in the buffer from the EZ1 kit yielded 90/110 (82%) positive specimens (P = 0.001) with an average Ct value of 27.4 (P = 0.001). The manual protocol yielded 100/110 (91%) positive specimens (P = 0.001) with an average Ct value of 30.33 (P = 0.001). However, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semi-automated protocol (P > 0.1 and P > 0.1).

Conclusion

Proteinase K digestion and glass powder crushing dramatically increase the extraction yield of archaea DNA from human stools. The semi-automated protocol described here was more rapid than the manual protocol and yielded significantly more archaeal DNA. It could be applied for extracting total stool DNA for further PCR amplification.

【 授权许可】

   
2013 Khelaifia et al.; licensee BioMed Central Ltd.

【 参考文献 】

• [1]Miller TL, Wolin MJ, de Macario EC, Macario AJ: Isolation of Methanobrevibacter smithii from human feces. Appl Environ Microbiol 1982, 43:227-232.
• [2]Miller TL, Wolin MJ: Methanosphaera stadtmaniae gen. nov. sp. nov: a species that forms methane by reducing methanol with hydrogen. Arch Microbiol 1985, 141:116-122.
• [3]Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M: Methanomassiliicocus luminyensis, gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. Int J Syst Evol Microbiol 2012, 62:1902-1907.
• [4]Gorlas A, Robert C, Gimenez G, Drancourt M, Raoult D: Complete genome sequence of Methanomassiliicoccus luminyensis, the largest genome of a human-associated Archaea species. J Bacteriol 2012, 194:4745.
• [5]Ferrari A, Brusa T, Rutili A, Canzi E, Biavati B: Isolation and characterization Methanobrevibacter oralis sp. nov. Curr Microbiol 1994, 29:7-12.
• [6]Dridi B, Raoult D, Drancourt M: Archaea as emerging organisms in complex human microbiomes. Anaerobe 2011, 17:56-63.
• [7]de Macario Conway E, Macario AJL: Methanogenic archaea in health and disease: a novel paradigm of microbial pathogenesis. Int J Med Microbiol 2009, 299:99-108.
• [8]DiBaise JK, Zhang H, Crowell MD, Krajmalnik-Brown R, Decker GA, Rittmann BE: Gut microbiota and its possible relationship with obesity. Mayo Clin Proc 2008, 83:460-469.
• [9]Zhang H, DiBaise JK, Zuccolo A, Kudrna D, Braidotti M, Yu Y: Human gut microbiota in obesity and after gastric bypass. P Natl Acad Sci 2009, 106:2365-2370.
• [10]Hans-Peter Horz GC: Methanogenic Archaea and oral infections - ways to unravel the black box. J Oral Microbiol 2011, 3:5940.
• [11]Lepp PW, Brinig MM, Ouverney CC, Palm K, Armitage GC, Relman DA: Methanogenic Archaea and human periodontal disease. P Natl Acad Sci USA 2004, 101:6176-6181.
• [12]Yamabe K, Maeda H, Kokeguchi S, Tanimoto I, Sonoi N, Asakawa S: Distribution of Archaea in Japanese patients with periodontitis and humoral immune response to the components. FEMS Microbiol Lett 2008, 287:69-75.
• [13]Schleper C, Jurgens G, Jonuscheit M: Genomic studies of uncultivated archaea. Nat Rev Microbiol 2005, 3:479-488.
• [14]van der Maarel MJEC, Sprenger W, Haanstra R, Forney LJ: Detection of methanogenic archaea in seawater particles and the digestive tract of a marine fish species. FEMS Microbiol Lett 1999, 173:189-194.
• [15]Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M: High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol. PLoS One 2009, 4:e7063.
• [16]Deuter R, Pietsch S, Hertel S, Müller O: A method for preparation of fecal DNA suitable for PCR. Nucleic Acids Res 1995, 23:3800-3801.
• [17]Mûller A, Stellermann K, Hartmann P, Schrappe M, Fâtkenheuer G, Salzberger B: A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens. Clin Diagn Lab Immun 1999, 6:243-246.
• [18]Bashalkhanov S, Rajora OP: Protocol: a high-throughput DNA extraction system suitable for conifers. Plant Methods 2008, 4:20. BioMed Central Full Text
• [19]Xin Z, Chen J: A high throughput DNA extraction method with high yield and quality. Plant Methods 2012, 8:26. BioMed Central Full Text
• [20]Scanlan P, Shanahan F, Marchesi J: Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis. BMC Microbiol 2008, 8:79. BioMed Central Full Text