A semi-automated protocol for Archaea DNA extraction from stools
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Published on: December 10, 2014
Saber Khelaifia1, Pierre-Yves Ramonet, Marielle Bedotto Buffet
1Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095, 13005 Marseille, France.
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.
