title: A semi-automated protocol for Archaea DNA extraction from stools.
abstract: 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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors: Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005 Marseille, France.
authors: Saber
authors: S
authors: Khelaifia
authors: Pierre-Yves
authors: PY
authors: Ramonet
authors: Marielle
authors: M
authors: Bedotto Buffet
authors: Michel
authors: M
authors: Drancourt
mesh_terms: D001105:Archaea / Q000235:genetics / Q000302:isolation & purification*; D001331:Automation; D001483:Base Sequence; D017931:DNA Primers; D019641:DNA, Archaeal / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D019641:DNA, Archaeal
references: Clin Diagn Lab Immunol. 1999 Mar;6(2):243-6
references: 10066661
references: J Bacteriol. 2012 Sep;194(17):4745
references: 22887657
references: Appl Environ Microbiol. 1982 Jan;43(1):227-32
references: 6798932
references: Plant Methods. 2008 Aug 01;4:20
references: 18673554
references: PLoS One. 2009 Sep 17;4(9):e7063
references: 19759898
references: Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6176-81
references: 15067114
references: Nat Rev Microbiol. 2005 Jun;3(6):479-88
references: 15931166
references: Anaerobe. 2011 Apr;17(2):56-63
references: 21420503
references: Arch Microbiol. 1985 Mar;141(2):116-22
references: 3994486
references: FEMS Microbiol Lett. 1999 Apr 1;173(1):189-94
references: 10220894
references: Nucleic Acids Res. 1995 Sep 25;23(18):3800-1
references: 7479018
references: Mayo Clin Proc. 2008 Apr;83(4):460-9
references: 18380992
references: J Oral Microbiol. 2011 Feb 23;3:
references: 21541092
references: Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2365-70
references: 19164560
references: Int J Med Microbiol. 2009 Feb;299(2):99-108
references: 18757236
references: Int J Syst Evol Microbiol. 2012 Aug;62(Pt 8):1902-1907
references: 22859731
references: Plant Methods. 2012 Jul 28;8(1):26
references: 22839646
references: FEMS Microbiol Lett. 2008 Oct;287(1):69-75
references: 18707623
references: BMC Microbiol. 2008 May 20;8:79
references: 18492229